Active pharmaceutical ingredients (APIs), which are incorporated in different formulations, i.e. creams, gels, foams, etc., are applied to the skin for a therapeutic effect. This therapeutic effect could either be required in the top layer of the skin (topical drug delivery) or deeper layers to reach the blood capillaries (transdermal drug delivery). Transdermal delivery avoids oral administration route limitations, such as first pass metabolism which is the rapid clearance of the drug in the gastrointestinal tract and degradation by enzymes. This delivery targets the drugs to skin sites, where there are significant advantages which include: improved patient compliance, a steady drug delivery state, less frequent dosing, adverse effects are minimal, it is less invasive and issues with the gastrointestinal absorption are avoided by eliminating the first pass metabolism (Perrie et al., 2012:392). This type of delivery is not free from limitations even though the skin can be employed for targeted drug delivery and is a readily available and large accessible surface area for adsorption of drugs. The most upper layer of the human skin, the stratum corneum, which is a watertight barrier, offers defence against hazardous exterior materials such as fungi, allergens, viruses and other molecules. This indicates the stratum corneum controls the drug penetration of most drugs to permeate the skin barrier (Lam & Gambari, 2014:27). Pravastatin is hydrophilic and is a 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor which inhibits cholesterol synthesis, a rate-limiting step, in the liver, thus decreasing the level of plasma low density lipoprotein cholesterol (LDL-C) (Heath et al., 1998:42). It can also slow the progression of atherosclerosis and can lower the incident of coronary events (Haria & McTavish, 1997:299). The first aim of the study is to deliver pravastatin transdermally into the blood circulation. Currently, pravastatin is only administered in oral dosages and can cause highly negative adverse effects such as myopathy and increased liver enzymes. This increase in liver enzymes causes hepatotoxicity and therefore would be ideal if pravastatin could be delivered transdermally, as the first pass metabolic effect would be nullified and adverse effects would decrease drastically (Gadi et al., 2013:648). Prof JW Wiechers‟ Delivery Gap Principle was designed in attempt to effectively enhance transdermal drug delivery. This Delivery Gap Principle was incorporated in the computer programme he developed known as “Formulating for Efficacy” (FFE™). The transdermal delivery of suggested APIs, which in this case is pravastatin, when incorporated into a formulation, may be optimised transdermally. The FFE™ computer programme suggests that the oil phase can be optimised, which in turn leads to better permeation through the skin to the target site (transdermal). The formula can be manipulated to reach desired polarity. The second aim of this in vitro study was to investigate the implementation of Wiechers‟ Delivery Gap Principle in a semi-solid dosage form, for the transdermal delivery of pravastatin sodium (2%). Six formulations, of which three were cream and three were emulgel formulations incorporated with pravastatin sodium (2%), were formulated. Each formulation had a different polarity, i.e. hydrophilic cream (HC) and emulgel (HE), lipophilic cream (LC) and emulgel (LE) and optimised cream (OC) and emulgel (OE). A high performance liquid chromatography (HPLC) method was developed and validated to analyse the concentration of pravastatin. Both the octanol-buffer distribution coefficient (log D) and the aqueous solubility of pravastatin were determined. For the API to permeate through the skin into the blood circulation, certain physicochemical properties are important and according to Naik et al (2000:321), there are specific ideal limits for the API in the formulations which include log D (1 to 3) and a aqueous solubility of >1 mg/ml. The aqueous solubility of 197.5 mg/ml in phosphate buffer solution (PBS) (pH 7.4) at a temperature of 32 °C indicated penetration was favourable (Naik et al., 2000:321), whilst the log D value of -0.703 indicated the API was unfavourable for skin penetration (Naik et al., 2000:321). Membrane release studies were conducted using a synthetic membrane to determine whether pravastatin was released from the six formulations each containing 2% pravastatin prior to diffusion studies with. The OE yielded the highest median flux value (7.175 μg/cm2.h), followed the by LE (6.401 μg/cm2.h), HE (6.355 μg/cm2.h), HC (5.061 μg/cm2.h), OC (4.297 μg/cm2.h) and lastly, LC (3.115 μg/cm2.h). By looking at the aforementioned data values, it was concluded that the emulgels performed better than the cream formulations when median flux values were compared. By using dermatomed excised female Caucasian skin, an execution of Franz cell diffusion studies were performed over a period of 12 h, followed by a tape-stripping experiment to determine which semi-solid formulation delivered pravastatin best on the skin and the results of the different polarity formulations were compared. The median amount per area which permeated through the skin after 12 h was as follows: the OE formulation (2.578 μg/cm2) depicted the highest median amount per area, followed by OC (1.449 μg/cm2), HC (0.434 μg/cm2), LE (0.121 μg/cm2), HE (0.055 μg/cm2) and lastly LC (0.000 μg/cm2). These results validate Wiechers` theory that when the oil phase is optimised, with regard to the same polarity as the skin, permeation will be enhanced (Wiechers, 2011). During both the membrane studies and the skin diffusion studies it was evident the emulgel formulations performed better and pravastatin permeated better than the cream formulations. When skin diffusion and membrane median data values were compared, it was evident in both the membrane release studies and the skin diffusion studies that OE yielded the highest median values and LC the lowest median values. It was clear that all six different formulations released pravastatin, but LC displayed no permeation into the systemic circulation (receptor phase). The data of the different polarity formulations which yielded the best results with regards to median concentrations within the stratum corneum-epidermis and epidermis-dermis, were identified and are: within the stratum corneum-epidermis, HE (1.448 μg/ml) yielded the highest median concentration pravastatin, followed by LE (1.301 μg/ml), LC (0.676 μg/ml), HC (0.505 μg/ml), OE (0.505 μg/ml) and lastly OC (0.400 μg/ml). As emulgels (hydrophilic) contain more water than creams (lipophilic), the penetration enhancement effect can be explained by hydration, since the water hydrated the skin leading the lipids to open and the stratum corneum to swell (Williams & Barry, 2004:606). Therefore more API could permeate into the skin. Within the epidermis-dermis the highest median concentration median was yielded by OE (0.849 μg/ml), followed by LC (0.572 μg/ml), HC (0.524 μg/ml), OC (0.355 μg/ml), HE (0.309 μg/ml) and lastly LE (0.138 μg/ml). Different polarity formulations permeating the viable epidermis could be a result of the solubility characteristics of the formulations. It contained both lipid properties (formulations contained oil content), leading to permeation through the stratum corneum and aqueous properties, which lead to diffusion into the underlying layers of the epidermis (Perrie et al., 2012:392). According to Perrie (2012:392), formulations that need to be delivered transdermally, must permeate through the lipophilic stratum corneum and thereafter the hydrophilic dermal layers to reach the blood circulation, which means formulations must consist of both lipophilic and aqueous solubility properties. When comparing the stratum corneum-epidermis (lipophilic) with the epidermis-dermis (more hydrophilic) and receptor phase (hydrophilic; systemic circulation), it is evident that all formulations had lipophilic and hydrophilic properties, as the API permeated through the stratum corneum and penetrated the deeper layers of the skin (viable epidermis) When all polarity formulations were compared, i.e. optimised, hydrophilic and lipophilic, it was observed that the optimised formulations depicted the highest median concentration values in the receptor phase (skin diffusion), but lowest median concentration in stratum corneum-epidermis, therefore the optimised formulation permeated best through the stratum corneum-epidermis. The reason for this could be that the optimised formulations had the same polarity as the skin (17, 8, 8), thus permeating through the skin to the receptor fluid more efficiently (Wiechers, 2011). It was observed that LC penetrated both stratum corneum-epidermis and epidermis-dermis, but did not permeate through the skin to the receptor fluid (systemic circulation), making it a good delivery vehicle for topical delivery. Overall when the emulgel and cream formulations are compared, according to their ability to deliver pravastatin transdermally, it is evident the pravastatin diffused more from the emulgel formulations than the cream formulations. This could be due to the fact that emulgels are more hydrophilic as they contain more water, resulting in the emulgels diffusing to the deeper layers of the skin (more hydrophilic viable epidermis) (Benson, 2005:28). All formulations contained not only aqueous (hydrophilic) but also lipid (lipophilic) solubility properties, therefore making it lipophilic enough to permeate the stratum corneum and hydrophilic enough to penetrate to deeper skin layers (viable epidermis) (Perrie et al., 2012:392). All formulations could still permeate the viable epidermis despite different polarities being used and all were appropriate candidates, although some were more suitable than others. The understanding from this study is that: * pravastatin could be delivered topically by all formulations, * the best formulation to reach the systemic formulation is the optimised emulgel, * the best formulation to deliver pravastatin topically is the hydrophilic emulgel. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Active pharmaceutical ingredients (APIs), which are incorporated in different formulations, i.e. creams, gels, foams, etc., are applied to the skin for a therapeutic effect. This therapeutic effect could either be required in the top layer of the skin (topical drug delivery) or deeper layers to reach the blood capillaries (transdermal drug delivery). Transdermal delivery avoids oral administration route limitations, such as first pass metabolism which is the rapid clearance of the drug in the gastrointestinal tract and degradation by enzymes. This delivery targets the drugs to skin sites, where there are significant advantages which include: improved patient compliance, a steady drug delivery state, less frequent dosing, adverse effects are minimal, it is less invasive and issues with the gastrointestinal absorption are avoided by eliminating the first pass metabolism (Perrie et al., 2012:392). This type of delivery is not free from limitations even though the skin can be employed for targeted drug delivery and is a readily available and large accessible surface area for adsorption of drugs. The most upper layer of the human skin, the stratum corneum, which is a watertight barrier, offers defence against hazardous exterior materials such as fungi, allergens, viruses and other molecules. This indicates the stratum corneum controls the drug penetration of most drugs to permeate the skin barrier (Lam & Gambari, 2014:27). Pravastatin is hydrophilic and is a 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor which inhibits cholesterol synthesis, a rate-limiting step, in the liver, thus decreasing the level of plasma low density lipoprotein cholesterol (LDL-C) (Heath et al., 1998:42). It can also slow the progression of atherosclerosis and can lower the incident of coronary events (Haria & McTavish, 1997:299). The first aim of the study is to deliver pravastatin transdermally into the blood circulation. Currently, pravastatin is only administered in oral dosages and can cause highly negative adverse effects such as myopathy and increased liver enzymes. This increase in liver enzymes causes hepatotoxicity and therefore would be ideal if pravastatin could be delivered transdermally, as the first pass metabolic effect would be nullified and adverse effects would decrease drastically (Gadi et al., 2013:648). Prof JW Wiechers‟ Delivery Gap Principle was designed in attempt to effectively enhance transdermal drug delivery. This Delivery Gap Principle was incorporated in the computer programme he developed known as “Formulating for Efficacy” (FFE™). The transdermal delivery of suggested APIs, which in this case is pravastatin, when incorporated into a formulation, may be optimised transdermally. The FFE™ computer programme suggests that the oil phase can be optimised, which in turn leads to better permeation through the skin to the target site (transdermal). The formula can be manipulated to reach desired polarity. The second aim of this in vitro study was to investigate the implementation of Wiechers‟ Delivery Gap Principle in a semi-solid dosage form, for the transdermal delivery of pravastatin sodium (2%). Six formulations, of which three were cream and three were emulgel formulations incorporated with pravastatin sodium (2%), were formulated. Each formulation had a different polarity, i.e. hydrophilic cream (HC) and emulgel (HE), lipophilic cream (LC) and emulgel (LE) and optimised cream (OC) and emulgel (OE). A high performance liquid chromatography (HPLC) method was developed and validated to analyse the concentration of pravastatin. Both the octanol-buffer distribution coefficient (log D) and the aqueous solubility of pravastatin were determined. For the API to permeate through the skin into the blood circulation, certain physicochemical properties are important and according to Naik et al (2000:321), there are specific ideal limits for the API in the formulations which include log D (1 to 3) and a aqueous solubility of >1 mg/ml. The aqueous solubility of 197.5 mg/ml in phosphate buffer solution (PBS) (pH 7.4) at a temperature of 32 °C indicated penetration was favourable (Naik et al., 2000:321), whilst the log D value of -0.703 indicated the API was unfavourable for skin penetration (Naik et al., 2000:321). Membrane release studies were conducted using a synthetic membrane to determine whether pravastatin was released from the six formulations each containing 2% pravastatin prior to diffusion studies with. The OE yielded the highest median flux value (7.175 μg/cm2.h), followed the by LE (6.401 μg/cm2.h), HE (6.355 μg/cm2.h), HC (5.061 μg/cm2.h), OC (4.297 μg/cm2.h) and lastly, LC (3.115 μg/cm2.h). By looking at the aforementioned data values, it was concluded that the emulgels performed better than the cream formulations when median flux values were compared. By using dermatomed excised female Caucasian skin, an execution of Franz cell diffusion studies were performed over a period of 12 h, followed by a tape-stripping experiment to determine which semi-solid formulation delivered pravastatin best on the skin and the results of the different polarity formulations were compared. The median amount per area which permeated through the skin after 12 h was as follows: the OE formulation (2.578 μg/cm2) depicted the highest median amount per area, followed by OC (1.449 μg/cm2), HC (0.434 μg/cm2), LE (0.121 μg/cm2), HE (0.055 μg/cm2) and lastly LC (0.000 μg/cm2). These results validate Wiechers` theory that when the oil phase is optimised, with regard to the same polarity as the skin, permeation will be enhanced (Wiechers, 2011). During both the membrane studies and the skin diffusion studies it was evident the emulgel formulations performed better and pravastatin permeated better than the cream formulations. When skin diffusion and membrane median data values were compared, it was evident in both the membrane release studies and the skin diffusion studies that OE yielded the highest median values and LC the lowest median values. It was clear that all six different formulations released pravastatin, but LC displayed no permeation into the systemic circulation (receptor phase). The data of the different polarity formulations which yielded the best results with regards to median concentrations within the stratum corneum-epidermis and epidermis-dermis, were identified and are: within the stratum corneum-epidermis, HE (1.448 μg/ml) yielded the highest median concentration pravastatin, followed by LE (1.301 μg/ml), LC (0.676 μg/ml), HC (0.505 μg/ml), OE (0.505 μg/ml) and lastly OC (0.400 μg/ml). As emulgels (hydrophilic) contain more water than creams (lipophilic), the penetration enhancement effect can be explained by hydration, since the water hydrated the skin leading the lipids to open and the stratum corneum to swell (Williams & Barry, 2004:606). Therefore more API could permeate into the skin. Within the epidermis-dermis the highest median concentration median was yielded by OE (0.849 μg/ml), followed by LC (0.572 μg/ml), HC (0.524 μg/ml), OC (0.355 μg/ml), HE (0.309 μg/ml) and lastly LE (0.138 μg/ml). Different polarity formulations permeating the viable epidermis could be a result of the solubility characteristics of the formulations. It contained both lipid properties (formulations contained oil content), leading to permeation through the stratum corneum and aqueous properties, which lead to diffusion into the underlying layers of the epidermis (Perrie et al., 2012:392). According to Perrie (2012:392), formulations that need to be delivered transdermally, must permeate through the lipophilic stratum corneum and thereafter the hydrophilic dermal layers to reach the blood circulation, which means formulations must consist of both lipophilic and aqueous solubility properties. When comparing the stratum corneum-epidermis (lipophilic) with the epidermis-dermis (more hydrophilic) and receptor phase (hydrophilic; systemic circulation), it is evident that all formulations had lipophilic and hydrophilic properties, as the API permeated through the stratum corneum and penetrated the deeper layers of the skin (viable epidermis) When all polarity formulations were compared, i.e. optimised, hydrophilic and lipophilic, it was observed that the optimised formulations depicted the highest median concentration values in the receptor phase (skin diffusion), but lowest median concentration in stratum corneum-epidermis, therefore the optimised formulation permeated best through the stratum corneum-epidermis. The reason for this could be that the optimised formulations had the same polarity as the skin (17, 8, 8), thus permeating through the skin to the receptor fluid more efficiently (Wiechers, 2011). It was observed that LC penetrated both stratum corneum-epidermis and epidermis-dermis, but did not permeate through the skin to the receptor fluid (systemic circulation), making it a good delivery vehicle for topical delivery. Overall when the emulgel and cream formulations are compared, according to their ability to deliver pravastatin transdermally, it is evident the pravastatin diffused more from the emulgel formulations than the cream formulations. This could be due to the fact that emulgels are more hydrophilic as they contain more water, resulting in the emulgels diffusing to the deeper layers of the skin (more hydrophilic viable epidermis) (Benson, 2005:28). All formulations contained not only aqueous (hydrophilic) but also lipid (lipophilic) solubility properties, therefore making it lipophilic enough to permeate the stratum corneum and hydrophilic enough to penetrate to deeper skin layers (viable epidermis) (Perrie et al., 2012:392). All formulations could still permeate the viable epidermis despite different polarities being used and all were appropriate candidates, although some were more suitable than others. The understanding from this study is that: * pravastatin could be delivered topically by all formulations, * the best formulation to reach the systemic formulation is the optimised emulgel, * the best formulation to deliver pravastatin topically is the hydrophilic emulgel. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

There is conflicting data on the effects of statins on cerebral vasospasm and clinical outcomes in aneurysmal subarachnoid hemorrhage (aSAH) patients. In this retrospective cohort study, patients were divided into those who received pravastatin (PRAV group) 40mg/d and those who did not (NP group). Data were analyzed using multivariate logistic regression. Eighty-one patients met inclusion criteria. There was a statistically significant decreased in the incidence of vasospasm in the PRAV group; however, this association did not retain significance after adjusting for WFNS, race, elevated WBC, and clipping (59% PRAV vs. 88% NP, p=0.08). There was no statistically significant difference in proportion of severe radiological vasospasm or mortality between groups. However, there was a trend towards a decreased mean length of stay (P=0.06) and a significantly higher proportion of survivors discharged to home in the PRAV group (P<0.0001). In conclusion, there was a trend towards a decrease in the incidence of vasospasm in the aSAH receiving pravastatin, but this trend did not achieve statistical significance after adjusting for potential confounders. Pravastatin was associated with other favorable clinical outcomes.

Por meio da técnica de microperfusão estacionária in vivo, túbulos proximais (TP) de ratos Wistar foram perfundidos com solução CTRL contendo ou não Sinva 100&#956;M ou Sil 10&#956;M para determinar a reabsorção de bicarbonato (JHCO3-). A perfusão de Sil diminuiu o JHCO3- em 20%, enquanto que Sinva incrementou em 19,31% o JHCO3- comparado com o CTRL. Já a Prav diminuiu em 15% o JHCO3- . Para testar se os efeitos foram dependents de NHE3, PTs foram perfundidos com CTRL, Sil, Sinva ou Prav mais S3226, um inibidor específico de NHE3. O JHCO3- remanescente (insensível à S3226) não foi diferente entre os grupos, mostrando que NHE3 foi modulado por Sil e Sinva. Já o efeito inibitório da Prav ocorre não apenas via NHE3 mas também via H+ ATPase. Para determinar se a via Rho está envolvida nestes efeitos, os PTs foram perfundidos com Y-27632 1&#956;M, inibidor de Rho GTPase. A perfusão com Y-27632 reverteu o efeito inibitório promovido tanto pelo Sil quanto pela Prav, demonstrando que o efeito inibitório destes fármacos sobre a atividade do NHE3 ocorre via Rho. Além disso, o efeito inibitório de Sil foi completamente abolido pelo inibidor de PKG dependente de GMPc KT5823 10-6 M. Para confirmar estes resultados, animais foram mantidos em gaiolas metabólicas por 24 h e tratados via oral com 7mg/Kg/dia de Sinva e 20mg/Kg/dia de Sil. Sil diminuiu a CE Na+, indicando que outros mecanismos de transporte podem estar envolvidos em alterações no manejo de Na+ pelos rins promovidas por Sil. Já a infusão de Prav em ratos Wistar pela jugular durante 30 min promoveu aumento da CE Na+, sugerindo que o fármaco age inibindo NHE3. / By means of stationary microperfusion, PT of Wistar rats were perfused with a CTRL solution with or without 100&#956;M Simva or 10&#956;M Sil to determine bicarbonate reabsorption (JHCO3-). Perfusion of Sil decreased JHCO3- by 20% and perfusion of Simva increased JHCO3- by 19,31% compared to CTRL. Prav decreased JHCO3- by 15%. To test if these effects were NHE3-dependent, PTs were perfused with CTRL, Sil, Simva or Prav plus 2&#956; M S3226, a specific NHE3 inhibitor. The reminiscent S3226-insensitive JHCO3- was not different among groups, showing that NHE3 was modulated by Sil or Simva. To determine if Rho was involved in these effects, the PTs were perfused with the Rho GTPase inhibitor Y-27632 (1&#956;M). Perfusion with Y-27632 reversed the inhibitory effect promoted by both Sil and Prav, demonstrating that the inhibitory effect of these drugs on NHE3 activity occurs via Rho. Furthermore, the inhibitory effect of Sil was completely abolished by either the protein kinase G (PKG) inhibitor dependent of cGPM KT5823 10-6 M. To confirm our results the animals were kept for 24 hours in metabolic cages and treated orally with 7mg/kg Sim or 20mg/kg Sil. The Sil decreased the Na+ excretion load, indicating that other transport mechanisms beyond the proximal tubule involved in the changes of handling of Na+ by the treatment with Sil. On the other hand, the infusion of Prav in Wistar rats by jugular for 30 min promoted an increase in Na+ CE, suggesting that the drug acts by inhibiting NHE3.

Glial-derived lipoparticles facilitate the transport of cholesterol and lipids between cells within the CNS and have been shown to support neuronal growth and synaptogenesis. Partial deafferentation of the hippocampus by unilateral entorhinal cortex lesioning (uECL) induces well-described cytoarchitectural reorganisation and reactive sprouting in the dentate gyrus (DG). Previous studies have demonstrated a dynamic regulation of cholesterol homeostasis in the hippocampus following deafferentation, and suggest that mechanisms facilitating cholesterol transport are important during reinnervation. Furthermore, there is growing evidence that statins, a family of cholesterol-lowering drugs which inhibit the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA-R), may confer neuroprotection following trauma. / The ATP binding cassette transporters (ABC) A1 and G1 assist the generation of lipoparticles by mediating cholesterol and phospholipid efflux to extracellular apolipoprotein E (APOE), the brain's primary lipoprotein. To examine a role for these transporters in the regulation of cholesterol efflux during synaptic remodelling, and the effects of low-dose pravastatin (a potent HMGCoA-R inhibitor) on such intercellular transport mechanisms, we measured the expression of ABCA1, ABCG1, APOE, apoE(LDL)R and HMGCoA-R in the hippocampus of saline and pravastatin treated mice over time following uECL. It is shown here that ABCA1 and not ABCG1 is up-regulated at the level of mRNA and protein expression, along with APOE, in the hippocampus during active regeneration (14DPL) as determined by histochemical analysis of acetylcholinesterase staining density in the DG. While pravastatin treatment was observed to differentially influence the expression of ABCA1 mRNA and protein over time, no effects on APOE or ABCG1 mRNA expression were observed following uECL. Additionally, HMGCoA-R mRNA expression was significantly down-regulated at 21 DPL in the deafferented hippocampus in pravastatin-treated animals. While the low-dose pravastatin treatment applied here was sufficient to inhibit HMGCoA-R activity in the liver, enzymatic activity was unaffected in the cortex. / These findings suggest that ABCA1 and not ABCG1 may be important in the APOE-mediated cholesterol recycling observed during the active phase of neural reinnervation in response to uECL. In addition, the results presented here suggest that the administration of clinically-relevant statin therapy may be sufficient to influence the regulation of cerebral cholesterol homeostasis following trauma in the adult mouse brain.

Orientador: Carlos Alan Candido Dias Junior / Resumo: Durante a gestação, diversas adaptações fisiológicas ocorrem no organismo materno para atender as necessidades nutricionais do feto em desenvolvimento. Entre essas adaptações, encontramos aumento do débito cardíaco e do volume sanguíneo, os quais são compensados pela drástica redução da resistência vascular sistêmica, promovendo a manutenção da pressão arterial materna nas gestações. O endotélio vascular apresenta um importante papel neste processo fisiológico por secretar fatores vasoativos como o óxido nítrico, um vasodilatador que se encontra aumentado em gestações saudáveis. Em algumas mulheres, falhas nesses mecanismos podem causar desordens hipertensivas gestacionais. A pré-eclâmpsia é uma desordem hipertensiva gestacional caracterizada pela elevação da pressão arterial (≥ 140 x 90 mmHg), após a 20ª semana gestacional. É frequentemente acompanhada de proteinúria e acomete 5-8% das gestantes. Atualmente, embora a fisiopatologia da pré-eclâmpsia seja desconhecida, estudos sugerem que sua ocorrência envolva migração deficiente de citotrofoblastos, culminando em má placentação e consequente isquemia/hipóxia placentária, responsável pela liberação de fatores bioativos promovendo danos às células endoteliais. A disfunção endotelial resulta na redução da biodisponibilidade de óxido nítrico, gerando as principais características da pré-eclâmpsia, como hipertensão materna e restrição de crescimento fetal. Recentes estudos apontam a importância da manutenção dos níveis de óxido n... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: During gestation, several physiological adaptations occur in the maternal organism to provide the nutritional demands of the developing fetus. Among these adaptations, we found increase in cardiac output and blood volume, which are compensated by the drastic reduction of systemic vascular resistance, promoting the maintenance of maternal blood pressure in gestations. Vascular endothelium plays an important role in this physiological process by secreting vasoactive factors such as nitric oxide, a vasodilator that is increased in healthy pregnancies. In some women, fails in these mechanisms may cause gestational hypertensive disorders. Preeclampsia is a gestational hypertensive disorder characterized by elevated blood pressure (≥ 140 x 90 mmHg) after the 20th gestational week. It is often accompanied by proteinuria and affects 5-8% of pregnant women. Currently, although the pathophysiology of preeclampsia is unknown, studies suggest that its occurrence involves poor migration of cytotrophoblasts culminating in poor placentation and consequent ischemia/placental hypoxia, responsible for the release of bioactive factors promoting damage to endothelial cells. Endothelial dysfunction results in the reduction of the bioavailability of nitric oxide leading to the occurrence of the main characteristics of preeclampsia, mainly maternal hypertension and fetal growth restriction. Recent studies point to the importance of maintaining levels of nitric oxide, which is associated with health... (Complete abstract click electronic access below) / Mestre

Preterm labour (PTL) is defined as labour before 37 completed weeks of gestation. Despite advances in medical research, PTL remains a major clinical problem. Preterm birth (PTB) rates range from approximately 5-18% worldwide. Importantly, PTB is the leading cause of childhood morbidity and mortality. PTL is difficult to predict and the aetiology is poorly understood but infection and inflammation are believed to be major factors. It has been suggested that the presence of intrauterine infection or inflammation may initiate the pathological, preterm activation of the inflammatory cascade associated with term labour. Therefore, PTL therapeutics should aim to inhibit these inflammatory pathways. Statins, 5-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, are potent inhibitors of cholesterol biosynthesis, which act on the mevalonate pathway. In addition to their lipid-lowering effects, statins also have anti-inflammatory and anti-contraction properties. The hypothesis of this thesis was that statins will prevent PTB by reducing inflammation. The aims of this thesis were firstly to investigate the effect of the statins, simvastatin and pravastatin, on inflammation and contractility in a pregnant human myometrial cell line. Secondly, to determine whether simvastatin and/or pravastatin can prevent PTB or improve neonatal outcome in a lipopolysaccharide (LPS)-induced mouse model of PTB. Myometrial cells were either co-treated with LPS and simvastatin/pravastatin, pretreated with simvastatin/pravastatin or treated with simvastatin/pravastatin post-LPS stimulation. The effect of statin treatment on the mRNA expression and the release of inflammatory mediators was then investigated. Simvastatin treatment reduced LPS-induced inflammation by both lowering the expression of pro-inflammatory mediators and increasing the expression of anti-inflammatory mediators. Pravastatin treatment did not alter the expression of inflammatory mediators following LPS stimulation. The effect of simvastatin on the contraction of myometrial cells was investigated by embedding the cells in rat tail collagen to form gels. As these are smooth muscle cells, basal contraction was observed causing the gel size to reduce. When LPS was introduced, this caused the gels to contract further than the vehicle treated gels. Simvastatin attenuated the contraction of the myometrial cells, both alone and in the presence of LPS. These effects were reversed by the addition of mevalonate pathway metabolites, mevalonate and geranylgeranyl pyrophosphate (GG-PP) but not by farnesyl pyrophosphate (F-PP). Simvastatin also lowered levels of phosphorylated myosin light chain (pMLC) in the myometrial cells, which is essential for smooth muscle contraction. Again, this effect was abolished by mevalonate and GG-PP but not F-PP. It is hypothesised that simvastatin attenuated myometrial cell contraction by inhibiting Rho isoprenylation by GG-PP, preventing Rho-associated kinase (ROCK) activation, which then prevented the phosphorylation of MLC. A mouse model of intrauterine LPS-induced PTB was utilised to investigate the effect of statin treatment on PTB and fetal survival. Mice received an intraperitoneal injection of pravastatin (10μg) or simvastatin (20μg or 40μg) on gestational day (D)16. This was followed by ultrasound-guided intrauterine injection of LPS (1μg) on D17 and another pravastatin/simvastatin treatment two hours later. When mice were treated with LPS, 77.8% of mice delivered preterm. When mice received LPS and 20μg simvastatin, 50% delivered preterm. However, when mice were treated with LPS and 40μg simvastatin, 40% delivered preterm, more pups were born alive and uterine pro-inflammatory mRNA expression was downregulated. Conversely, pravastatin did not prevent PTB or improve the percentage of live born pups. In summary, simvastatin treatment exerted anti-inflammatory and anti-contraction effects on human myometrial cells in vitro. The anti-contractile properties were likely due to the inhibition of the Rho/ROCK pathway. Furthermore, in our LPS-induced mouse model of PTB, fewer mice delivered preterm with simvastatin treatment, simvastatin attenuated LPS-induced pup mortality and reduced uterine inflammatory gene expression. These results suggest that statin therapy may be a novel treatment for PTL.

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Agradecemos a compreensão. on 2016-08-23T18:18:39Z (GMT) / Submitted by MARIANA MAURO null (mamaurofarm@gmail.com) on 2016-08-23T21:13:02Z No. of bitstreams: 1 DISSERTAÇÃO FINAL.pdf: 2164316 bytes, checksum: 594c78c0b6e6709fd92f1b941f2b7d32 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-24T15:04:03Z (GMT) No. of bitstreams: 1 mauro_m_me_arafcf.pdf: 2164316 bytes, checksum: 594c78c0b6e6709fd92f1b941f2b7d32 (MD5) / Made available in DSpace on 2016-08-24T15:04:03Z (GMT). No. of bitstreams: 1 mauro_m_me_arafcf.pdf: 2164316 bytes, checksum: 594c78c0b6e6709fd92f1b941f2b7d32 (MD5) Previous issue date: 2016-07-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A exposição ocupacional a agentes químicos é potencialmente capaz de modificar a disposição cinética ou o metabolismo de fármacos, principalmente em função da indução ou inibição de enzimas do sistema citocromo P450. Entretanto, estudos que mostram o efeito da exposição ocupacional a agentes químicos na atividade de transportadores de fármacos são escassos. O tolueno está presente no ambiente de trabalho principalmente na forma de vapor. Neste ambiente, o solvente é absorvido principalmente por inalação. O objetivo do estudo é avaliar a influência da exposição inalatória ao tolueno na atividade in vivo do polipeptídeo transportador de ânions orgânicos da família Oatp em ratos, empregando como fármaco marcador a pravastatina. Ratos machos Wistar (n=6 por tempo de coleta, por grupo) foram expostos ao tolueno (85 mg/m3) por inalação ou ao ar em câmara de exposição apenas pelo nariz por 6 horas/dia, 5 dias/semana, 4 semanas. Ao final do período de exposição, os animais receberam dose única de pravastatina (20 mg/kg) por via oral. Amostras seriadas de sangue foram coletadas até 8 horas após a administração de pravastatina. As concentrações plasmáticas do fármaco foram avaliadas por cromatografia líquida de alta eficiência com detecção por espectrometria de massas (LC-MS). O método de análise da pravastatina por LC-MS mostrou detectabilidade, precisão e exatidão compatíveis com a aplicação em estudos de disposição cinética após dose única em ratos. As áreas sob as curvas de concentração plasmática versus tempo (ASC) foram calculadas do tempo zero a infinito com base na Quadratura de Gauss-Laguerre. As concentrações de pravastatina correspondentes aos tempos avaliadas foram estimadas por interpolação polinomial. O clearance total aparente (CL/F) foi calculado a partir da equação: CL/F = dose/ASC0-∞. Na avaliação da disposição cinética da pravastatina, os animais não expostos ao tolueno apresentaram ASC0-∞ de 726 ng h/mL e Cl/F de 49,1 L/h kg. Não houve diferença estatística na ASC0-∞ ao comparar os animais expostos (ASC0-∞: 681,8 ng h/mL) e não expostos ao tolueno. Os resultados do estudo permitem concluir que a atividade de transportadores Oatp, avaliada pelo uso da pravastatina como fármaco marcador, não foi alterada após a exposição ao tolueno por inalação a 85 mg/m3, 6 horas/dia, 5 dias/semana, 4 semanas em ratos. / Occupational exposure to chemical agents is potentially able to modify the kinetic disposition of drug metabolism, mainly due to the induction or inibition of cytochrome P450 enzymes. However, studies investigating the influence of occupational exposure to chemicals on the variability in drug response are rare. Toluene is present in the working environment particularly in vapor form. In this environment, the toluene is mainly absorbed by inhalation. Considering that, the aim of this study was evaluate the influence of inhalation to toluene in the in vivo activity of the polypeptide transporter organic anion (Oatp) family transporters in rats, using pravastatin as a probe drug. Male Wistar rats (n=6, for each sampling time) were exposed to toluene (85 mg / m3) by inhalation or air in a nose only exposure system for 6 hours/day, 5 days/week, during 4 weeks. After 4 weeks of exposure, animals received a single dose of pravastatin (20 mg/kg) orally. Serial blood samples were collected up to 8 hours after administration of pravastatin. Plasma concentrations of pravastatin were measured by high-performance liquid chromatography with detection by mass spectrometry (LC-MS). The analysis method of pravastatin by LC-MS showed detectability, precision and accuracy compatible with the application in kinetic disposition studies after single doses in rats. The areas under the plasma concentration versus time (AUC) were calculated by zero to the infinity by the Gauss-Laguerre Quadrature. The concentrations corresponding to the times were estimated by polynomial interpolation. The total clearance apparent (CL/F) was calculated from the equation: CL/F = dose/AUC0-∞. In assessing the kinetics of pravastatin disposition, animals not exposed to toluene showed AUC0-∞ of 726 ng h/mL and CL/F of 49.1 L/h kg. There was no statistical difference in AUC0-∞ to compare the exposed animals (AUC0-∞: 681.8 ng h/mL) and not exposed to toluene. The results of this study showed that Oatp family transporters activity, assessed by the use of pravastatin as a marker drug, was not altered after exposure to toluene by inhalation at 85 mg/m3 6 hours/day, 5 days/week and for 4 weeks in rats. / CNPq: 448803/2014-3

Orientador: Eros Antonio de Almeida / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T18:51:46Z (GMT). No. of bitstreams: 1 Taraborelli_Simone_M.pdf: 7158008 bytes, checksum: d0f4a8ee6fd688737f4070fd4a3082d6 (MD5) Previous issue date: 2011 / Resumo: Objetivo: Este estudo teve por objetivo avaliar a ação da pravastatina em ratos infartados normocolesterolêmicos em relação aos lípides plasmáticos, colesterol e peroxidação lipídica tecidual e função endotelial. Métodos: Foram utilizados cinquenta ratos wistar machos com três meses de idade e pesando entre 300 a 400 gramas, divididos em cinco grupos (N=10) e divididos nos seguintes grupos: grupo ratos controle (G1), grupo ratos sham (G2), grupo ratos com infarto experimental do miocárdio (G3), grupo infarto experimental do miocárdio e pravastatina sete dias após o infarto (G4) e grupo infarto experimental do miocárdio e pravastatina sete dias antes e acrescido de sete dias após infarto experimental do miocárdio (G5). A pravastatina foi administrada na dose de 7,5mg/kg/dia via gavagem. Os grupos G3, G4 e G5 foram submetidos a infarto do miocárdio por meio da ligadura da coronária descendente anterior. No final do experimento de cada grupo ocorreu a canulação da artéria carótida para verificação da pressão arterial. Os animais foram sacrificados mediante anestesia com tiopental sódico 30 mg/kg e, após, foi realizado o estudo da função endotelial, dosagem dos lípides plasmáticos, colesterol e peroxidação lipídica tecidual. Para análise dos resultados utilizou-se o teste de Man Whitney e análise de variância. Resultados: Não houve diferenças significativas entre os grupos quando observado os valores de glicose e triglicérides. O colesterol total no grupo G4 foi significantemente menor quando comparado aos demais grupos (p<0,05). Em relação a HDL - colesterol houve significância quando comparado o grupo G5 aos grupos G3 e G4. A LDL - colesterol apresentou-se significantemente menor quando comparado os grupos G4 e G5 ao grupo G3. O colesterol tecidual nos grupos G4 e G5 foi significantemente menores quando comparado ao grupo G3. A diferença significativa em relação à peroxidação lipídica ocorreu no grupo G4 quando comparado ao grupo G2. O relaxamento dependente do endotélio foi significante maior quando comparado os grupos G4 e G5 aos grupos G1, G2 e G3. Em relação à pressão arterial a significância ocorreu entre os grupos G3 e G4. Conclusão: A pravastatina, quando administrada em ratos normocolesterolêmicos com infarto do miocárdio, diminuiu o colesterol total, LDL - colesterol, colesterol tecidual, peroxidação lipídica e aumentou HDL - colesterol. Quanto à função endotelial, a pravastatina quando administrada antes, durante ou após o infarto resultou em melhora da disfunção endotelial / Abstract: Objective: To evaluate pravastatin action in the experimental myocardial infarct regarding to the endothelial function, plasmatic lipid, tissue lipidic peroxidation and cholesterol in infarcted normocholesterolemic rats. Materials and Methods: Fifty wistar rats were used divided in 5 groups (n=10): control group (G1), sham group (G2), infarct group (G3), infarct group with pravastatin seven day after the infarct (G4) and infarct group with pravastatin 7 day before and 7 day after the infarction (G5). Pravastatin was administrated 7,5mg/Kg/day via gavage. The groups G3, G4 and G5 were infarcted through the anterior descendent coronary tying. The animals were sacrificed by means of anesthesia with sodium thiopental 30mg/Kg and after this the endothelial function (Rmax), total cholesterol dosage (CT), triglycerides (TG), high density lipoprotein (HDL - c), low density lipoprotein (LDL - c), tissue lipidic peroxidation (MDA) and (Cte) cholesterol were performed. In order to do the statistical analysis the Man Whitney Test and variance analysis were used (p? 0,05 significance). Results: There was no significant difference among the groups when compared the glucose values and triglycerides. The total cholesterol G4 groups it was smaller significant when compared to the other groups (p<0, 05). In relation to HDL - c there was significance when compared the G5 group to the G3 and G4 group. LDL - c presented - if smaller significantly when compared the G4 and G5 groups to the G3 groups. The tissue cholesterol in the groups G4 and G5 there was smaller significant when compared to G3 group. The significant difference in relation to lipidic peroxidation it happened in the group G4 when compared to group G2. The dependent relaxation of the endothelium it was significant larger when compared the G4 and G5 groups to the G1, G2 and G3 groups. In relation to blood pressure the significant happened between the G3 and G4 groups. Conclusion: Pravastatin when administered in normocholesterolemic with myocardial infarct, it reduces the total cholesterol, LDL - c, tissue cholesterol, lipidic peroxidation and it increased HDL - c. As the function endothelium, the pravastatin when administered in normocholesterolemic rats with myocardial infarct, before, during or after the infarct of the myocardium it resulted in improvement of the endothelium dysfunction / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

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