Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

During repetitive stimulation of motor nerve terminals, mitochondrial Ca2+ uptake limits increases in free cytosolic [Ca2+] and helps ensure faithful neuromuscular transmission. Changes in cytosolic [Ca2+] and in mitochondrial [Ca2+] as well as changes in mitochondrial membrane potential (Psi m) were studied in mouse motor nerve terminals using Ca2+ sensitive indicator and potentiometric dyes, respectively. Trains of action potentials (APs) at 50 to 100 Hz produced a rapid increase in mitochondrial [Ca2+] followed by a plateau which usually continued beyond the end of stimulation. After stimulation, mitochondrial [Ca2+] decayed back to baseline over the course of tens of seconds to minutes. Increasing the Ca2+ load delivered to the terminal by increasing the number of stimuli (500-2000), increasing bath [Ca2+], or prolonging the AP with 3,4-diaminopyridine (3-4, DAP, 100 micromolar), prolonged the post-stimulation decay of mitochondrial [Ca2+] without increasing the amplitude of the plateau. Inhibiting openings of the mitochondrial permeability transition pore with cyclosporin A (5 micromolar) had no significant effect on the decay of mitochondrial [Ca2+]. Inhibition of the mitochondrial Na+-Ca2+ exchanger with CGP-37157 (50 micromolar) dramatically prolonged the post-stimulation decay of mitochondrial [Ca2+], reduced post-stimulation residual cytosolic [Ca2+], and reduced the amplitude of end-plate potentials evoked after the end of stimulation. Stimulation-induced mitochondrial Ca2+ uptake resulted in Psi m depolarizations that were small or undetectable at near-physiological temperatures (~30 degrees C). Their amplitude became larger at lower temperatures (~20 degrees C), or when AP duration was increased with 3,4-DAP (20 micromolar). Psi m depolarizations were inhibited by lowering bath [Ca2+] or by blocking P/Q-type Ca2+ channels with omega-agatoxin (0.3 micromolar). Partial inhibition of complex I of the electron transport chain (ETC) with rotenone (50 nM) increased the amplitude of stimulation-induced Psi m depolarizations. These findings suggest that: (1) Ca2+ extrusion from motor terminal mitochondria occurs primarily via the Na+-Ca2+ exchanger and helps sustain post-tetanic transmitter release, and (2) that the depolarization of Psi m that accompanies Ca2+ uptake is limited by accelerated proton extrusion via the ETC.

This study seeks to understand the scope and types of public relations practices in Japan, how public relations practices of Japanese and foreign companies differ, how media respond differently to the public relations activities of Japanese and foreign companies, and how "foreignness" affects public relations activities of multinational enterprises in Japan. The sole academic journal in the field of public relations in Japan and the single commercially published public relations industry magazine were submitted to an empirical content analysis. Additionally, three cases of contest for corporate control, two of which were between an American activist fund and a Japanese company, and one between a Japanese investment fund and a Japanese company, were analyzed using a case study approach. Press releases and newspaper articles about these cases were also content analyzed. The results show that among public relations activities, crisis category appeared the most frequently in the public relations industry journal and corporate communication category appeared the most frequently in the public relations academic journal. The analysis of the cases shows that the American fund at first faced problems communicating with its stakeholders but improved its communication activities in subsequent years with better results. The content analysis of the press releases shows that American fund uses more quotes and persuasive messages in the press releases and Japanese newspapers do not treat domestic and foreign entities differently. The overall results confirm the importance of culture in public relations practices.

Polymeric excipients are generally the least well-characterized components of pharmaceutical formulations. The aim of this dissertation work is to facilitate the quality-by-design (QbD) approach to pharmaceutical formulation and manufacturing by evaluating the inter-grade and inter-batch variability of pharmaceutical-grade polymeric excipients. Sodium alginate, a widely used polymeric excipient, was selected for evaluation using appropriate analytical methods and test conditions, especially rheological methods. The materials used were six different grades of sodium alginate and an additional ten batches of one of the grades. <br>To compare the six grades, steady shear measurements were conducted on solutions at 1, 2, and 3% w/w, consistent with their use as thickening or binding agents. Small amplitude oscillation (SAO) measurements were conducted on sodium alginate solutions at higher concentrations (4-13% w/w) corresponding to their use in controlled release matrices. In order to compare the ten batches of one grade, steady shear and SAO measurements were performed on their solutions at 2% w/w and 8% w/w, respectively. Results show that rheological properties of sodium alginate solutions are influenced by both molecular weight and chemical composition of sodium alginate. ¡§One-point¡¨ apparent viscosity data obtained at one low concentration and one shear rate is not representative of the complex rheological behavior of various grades of sodium alginate solutions at higher concentrations or other shear rates. The potential interchangeability of these different grades used as thickening or binding agents could be established by comparing the apparent viscosities of their solutions as a function of both alginate concentration and shear conditions. For sodium alginate used in controlled release formulations, both steady shear (at one low concentration, e.g., 2% w/w) and SAO measurements (at one high concentration indicative of polymer gel state, e.g., 8% w/w) are recommended to be performed on sodium alginate solutions to ensure interchangeability. Furthermore, among batches of the same grade, significant differences in rheological properties were observed, especially at the high solution concentration (i.e., 8% w/w). In summary, inter-grade and inter-batch variability of sodium alginate can be determined using steady shear and SAO methods. <br>The influence of inter-grade and inter-batch variability of sodium alginate on the functionality of sodium alginate used in matrix tablets was investigated with a focus on compression properties, swelling, erosion behavior of alginate matrix tablets, and drug release from matrix tablets. The compression behavior of four grades and three batches of sodium alginate were studied by compaction energetics, out-of-die Gurnham, and out-of-die Heckel analysis. It was found that sodium alginates deform less plastically than microcrystalline cellulose (MCC PH102) but similar to lactose anhydrous. Sodium alginates also demonstrate more elastic deformations during compression than both MCC PH102 and lactose anhydrous. Compacts prepared from multiple batches of the same grade varied in porosity. The same tensile strength of compacts can be achieved by compressing the multiple batches to the same porosity. <br>Sodium alginate tablets undergo both swelling and erosion in water. Grades with substantially higher apparent viscosities at low solution concentration exhibit a higher percentage of water uptake and a low percentage of erosion. Those batches not significantly different in their apparent viscosities at low solution concentration but significantly different in viscoelasticity at high solution concentrations do demonstrate significant differences in their swelling and erosion behavior. Acetaminophen release from sodium alginate matrix tablets prepared from the four grades and three batches can be well described by a zero-order equation. Significant differences in release profile were observed among various grades and batches. <br>In conclusion, the inter-grade and inter-batch variability of sodium alginate has a significant influence on the swelling, erosion, and drug release behavior of sodium alginate matrix tablets. Apparent viscosities of sodium alginate solution at low concentration alone are not sufficient to predict the functionality of sodium alginate used in matrix tablets. Viscoelastic properties of sodium alginate solutions at high concentrations indicative of polymer gel state are appropriate to be characterized. <br>Further study was conducted to determine whether sodium alginate solutions¡&brkbar; rheological parameters are relevant to sodium alginate¡&brkbar;s use in the formulation of calcium alginate gels. Among the grades with similar guluronic acid percentage (%G), there is a significant correlation between gel fracture force and apparent viscosity. However, the results for the partial correlation analysis for all six grades of sodium alginate show that gel fracture force is significantly correlated with %G, but not with the rheological properties of the sodium alginate solutions. Studies of the ten batches of one grade of sodium alginate show that apparent viscosities of their solutions do not correlate with gel fracture force while tan <em>f</em>Ô values are significantly, but minimally, correlated to gel fracture force. Inter-batch differences in the rheological behavior for one specific grade of sodium alginate are insufficient to predict the corresponding calcium alginate gel's mechanical properties. <br>In summary, rheological methods, including steady shear and small amplitude oscillation, are able to identify the inter-grade and inter-batch variability of sodium alginate. Inter-grade and inter-batch variability of sodium alginate could lead to substantial differences in the functionality of sodium alginate in matrix tablets and in calcium alginate gels. Rheological properties of sodium alginate in solution are suggestive of its functionality as thickeners, or as controlled release agent. However, rheological properties of sodium alginate in solution do not seem to be sufficient to predict the mechanical properties of the corresponding calcium alginate gels. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences / Pharmaceutics / PhD / Dissertation

Le développement de formes à libération prolongée destinées à ladministration pulmonaire est un domaine qui a, jusquà présent, été relativement peu étudié mais pour lequel il y a actuellement un intérêt croissant. Le but de notre travail est de développer une forme destinée à ladministration par inhalation qui libérerait de façon prolongée un agent bronchodilatateur. Dans le cadre de ce travail, les microparticules lipidiques solides (MLS) ont été choisies comme véhicule en vue de lobtention dune libération prolongée. Les MLS présentent en effet de nombreux avantages en termes de coûts de production, de stabilité et de biocompatibilité comparativement à dautres systèmes microparticulaires. Le salbutamol, principe actif ß2-mimétique choisi initialement pour cette étude, nétant pas suffisamment lipophile pour sincorporer de façon efficace dans les MLS, un dérivé plus lipophile du salbutamol, lacétonide de salbutamol (AS) a été synthétisé. Les caractéristiques physico-chimiques de lAS ont été déterminées, sa stabilité a été évaluée et des méthodes de dosage ont été mises au point. Des MLS vierges (non chargées en AS) ont tout dabord été produites après optimisation des paramètres de fabrication en vue dobtenir une taille adéquate pour ladministration par inhalation. Des études de tolérance au niveau pulmonaire effectuées in vivo sur des rats ont montré la biocompatibilité de ces MLS. Lactivité pharmacologique de lAS a été évaluée à la fois par des essais ex vivo de bronchodilatation sur organes isolés ainsi que par des essais daffinité (binding) envers les récepteurs ß1 et ß2-adrénergiques. Ces études nont malheureusement pas permis de conclure avec certitude quant à léventuel effet ß2-mimétique de lAS. Cependant, en raison de son caractère lipophile, lAS sera utilisé comme molécule modèle tout au long du processus de développement. LAS a donc été incorporé dans les MLS et les paramètres de production ont été étudiés et fixés par la méthodologie des plans dexpérience en vue doptimiser le pourcentage de particules possédant un diamètre géométrique convenant à ladministration par inhalation. La concentration en AS naffectant pas de façon significative la taille des MLS, celles-ci pourront être produites en utilisant la concentration en AS désirée. La caractérisation de ces MLS par microscopie électronique a montré que, lorsque la charge théorique initiale augmente, des cristaux dAS sont observés à lextérieur des MLS. Des essais de libération menés in vitro dans un premier temps puis ex vivo (en présence de fragments de poumons de porcs) ont permis de montrer une prolongation de la libération de lAS à partir des MLS comparativement à la libération à partir de mélanges physiques de MLS vierges et dAS. Ces résultats montrent la capacité des MLS possédant une taille pour administration pulmonaire, à libérer de façon prolongée la molécule qui y est incorporée. Cette libération est dautant plus prolongée que la charge en AS diminue. La présence denzymes pulmonaires na cependant pas modifié la cinétique de libération. Des poudres pour inhalation à base de MLS à 5% en AS ont été formulées en utilisant différents excipients porteurs et compétiteurs en différentes concentrations relatives. Les fractions respirables mesurées in vitro sont, dans le meilleur des cas, égales à 15%. Cependant, les MLS à 5% en AS administrées seules ont une fraction respirable proche de 25%. Leurs propriétés découlement savérant acceptables, il peut être envisagé dadministrer les MLS telles quelles en tant que poudre pour inhalation./ The sustained release of drugs for pulmonary delivery is a research field which has been so far rather unexploited but which is currently becoming increasingly attractive. The aim of this research work is to develop a pulmonary delivery system which will be able to sustain the release of a bronchodilator agent. Therefore, solid lipid microparticles (SLMs) were chosen in order to provide a sustained release to its incorporated substance. Indeed, this kind of drug carrier offers many advantages. In comparison with other microparticulate dosage forms, SLMs production costs are relatively low, they are physiologically compatible and their physical stability is well established. Salbutamol, a well-known short-acting ß2-adrenergic receptor agonist, was initially chosen for this study but this molecule proved to be not lipophilic enough to be efficiently incorporated into SLMs. Thats the reason why salbutamol acetonide (SA) was synthetized from salbutamol in order to get a more lipophilic molecule and thereby to increase the incorporation into SLMs. Then, the physico-chemical properties of SA were characterized, its stability was studied and chromatographic assays were developed. Drug free SLMs were produced using manufacturing parameters which were optimized in order to get particles with a suitable range of size for pulmonary administration. Tolerance studies were then carried out in vivo on rats to check SLMs biocompatibility in the respiratory tractus. Ex vivo tests using isolated organs were carried out in order to investigate the bronchodilating activity of SA. The obtained results were completed with a binding study to evaluate the affinity between SA on the one hand and ß1 and ß2-adrenergic receptors on the other hand. Unfortunately those studies didnt allow us to conclude about the possible ß2-mimetic activity of SA. Owing to its lipophilic character, SA will be used all along this research work as a model molecule for the development of SLMs as sustained release system for pulmonary delivery. SA was then incorporated into SLMs: the production parameters were studied using the methodology of experimental design in order to optimize the percentage of particles with a suitable diameter for pulmonary administration. It has been noticed that SA concentration does not affect significantly the particle size. So SLMs can be produced using the pre-established production parameters whatever the desired SA concentration. The characterization of the obtained SLMs-SA by scanning electron microscopy showed especially that SA crystals appear outside of the particles when the theoretical drug loading increases. Drug release studies were carried out both in vitro and ex vivo i.e. using fragments of porcine pulmonary tissues. These studies showed that SA release from SLMs is sustained in comparison with SA release from physical mixtures of drug free SLMs and SA. The obtained results tend to prove that produced SLMs are suitable carriers in order to get a sustained release of the incorporated substance. It has also been noticed that the release rate increases when the drug loading increases. Concerning the ex vivo studies, it may be concluded that the presence of pulmonary enzymes does not modify SA release profiles. Inhalation powders containing SLMs with 5% SA were finally developed using different carrier excipients and ternary agents at different relative concentrations. Respirable fractions were determined in vitro and proved to be at best equal to 15%. However, SLMs 5% SA have also been administered alone without any additional excipients. In this case, the obtained respirable fraction is close to 25%. Seeing that the flowability of SLMs 5% SA appeared to be acceptable, they could be administered just as they are as inhalation powder.

<p>The focus of this study is to synthesize a novel hinged polymer actuator. The linking molecule (hinge) is phenazine with interconnected dipyrrole units.</p>

Granulocyte adhesion and release of their granule proteins are key steps during selective accumulation of a certain cell to an inflammatory site. Eosinophils are specifically recruited to sites of allergic inflammation and parasitic infection, whereas neutrophil influx predominates in bacterial infection and rheumatoid arthritis. A simple, reliable and convenient method was developed for the measurement of granulocyte adhesion and release of granule proteins by using the normal population of granulocytes. The design allows simultaneous quantitative assessment of eosinophil and neutrophil adhesion to proteins and degranulation. Using this method, manganese ions (Mn2+) induced a higher level of eosinophil adhesion to fibronectin, fibrinogen and albumin as compared with neutrophils. PMA induced comparable levels of eosinophil and neutrophil adhesion. F-MLP stimulated a rapid, short-term adhesion of neutrophils to fibrinogen. In the same conditions PMA alone stimulated a dose-dependent release of ECP from cells that adhered to both fibronectin and fibrinogen. Meanwhile, Mn2+ amplified the release of ECP induced by PMA. Furthermore, release of ECP was shown to be associated with cell death. PMA, in combination with Mn2+, induced a marked release of ~ 80%of the intracellular content of lactoferrin and HNL in neutrophils. PMA or f-MLP alone induced 30-40% release of lactoferrin and HNL. A maximal release of MPO of 15-20% was obtained from neutrophils stimulated by PMA and Mn2+. Release of lactoferrin and HNL showed a significant negative relationship to the viability of cells. Stimulated by PMA, eosinophils from pollen-atopic patients during early pollen season displayed a markedly enhanced adhesion and release of ECP of eosinophils compared with eosinophils from the references. Priming with IL-5 caused a significantly higher adhesion and release of ECP by eosinophils in response to PMA. GM-CSF priming enhanced eosinophil adhesion in response to PAF and PMA plus Mn2+, but did not enhance the release of ECP. In conclusion, the assay allows a simple quantification of eosinophil and neutrophil adhesion, as well as degranulation by using the normal population of granulocytes. Cellular adhesion plays an important role in the regulation of both eosinophil and neutrophil degranulation, but adhesion and degranulation can be induced separately.

The kidneys play a vital role in the maintenance of extracellular fluid and electrolyte balance and blood pressure. Adenosine, acting through the adenosine A1-receptor (A1R), and nitric oxide have been implicated in several of the regulatory mechanisms in the kidney. The A1R has been found to be present in the renal vasculature, primarily in the afferent arterioles, and in the proximal tubules. The tubuloglomerular feedback mechanism (TGF) is an important regulator of renal vascular tone and glomerular filtration rate. The aim of these investigations was to further elucidate the role of adenosine, acting through the A1R. Investigations on adenosine’s renal effects were performed on transgenic mice lacking the A1R. TGF response, elicited by increased distal salt load, was completely abolished in the A1R knockout (A1R -/- ) mice. Basal plasma-renin levels were found to be ~2-fold higher in the A1R -/- compared to the A1R wild-type (A1R+/+) mice. However, salt intake induced inverse changes in plasma-renin levels, indicating that adenosine tonically inhibits macula densa stimulated renin release. Anesthetized and conscious A1R -/- mice, measured telemetrically, had an increased blood pressure, which could be due to the increased plasma-renin levels. Despite the high plasma-renin levels, increased urinary sodium excretion was also observed in the A1R -/- animals. Ischemia caused a decrease in renal function in both A1R+/+ and A1R -/- mice. Ischemic preconditioning protected the A1R+/+ mice from subsequent ischemic episode but had no protective effect on the A1R -/- mice. Acute extracellular volume expansion greatly attenuates TGF sensitivity, thus facilitating the elimination of excess fluid. Acute inhibition of nNOS in volume-expanded rats was found to re-establish the attenuated TGF response caused by acute extracellular volume expansion. The results show that adenosine, acting through the A1R, plays an important role in mediating TGF response and consequently, regulating renin release, blood pressure, electrolyte balance and other vital renal mechanisms.

Impulses coming from peripheries are modified in the spinal cord and transmitted to the brain. Several neurotransmitters have been involved in the processing of impulses in the spinal dorsal horn. Acetylcholine (ACh) is one of many neurotransmitters involved in the regulation of nociception in the spinal cord. In this study we investigated the role of nicotinic, muscarinic, serotonergic and GABA receptors in the regulation of spinal ACh release since these receptors are reported to be involved in spinal nociceptive processes. Different receptor ligands were infused intraspinally via microdialysis and the spinal ACh release was measured by on-line HPLC. Receptor-ligand binding studies were performed with spinal cord homogenates as well as receptors expressed in cells. In the first study, we found that nicotine and some of the nicotinic antagonists used increased ACh release suggesting that spinal ACh release is regulated by different nAChRs. Nicotine and nicotinic agonists may act on different types of receptors with different affinity to produce the observed net effect of increased ACh release. We propose the possibility of an involvement of three different nicotinic receptor subtypes in the regulation of spinal ACh release. The effect of epibatidine, which is regarded as a nicotinic agonist, on muscarinic receptors was investigated in the second study. We propose that epibatidine, in μM concentrations, is a partial muscarinic receptor agonist that may interact with spinal muscarinic receptors to increase ACh release. The dual action on both nAChRs and mAChRs may explain the potent analgesic effect observed after intra-spinal epibatidine administration. In the third study, we investigated the role of serotonin receptor involvement in ACh release control. The results suggest that only 5-HT1A and 5-HT2A receptors are involved in spinal ACh release. Considering current knowledge, the most probable location of 5-HT2A receptors is on cholinergic neurones. On activation of the 5-HT2A receptors the cellular excitability of cholinergic neurones is increased which results in an increasing ACh release. The 5-HT1A receptors might be located on cell bodies of GABA neurones which inhibit the firing rate of the GABA neurones when activated by serotonin. In the fourth study, we investigated the GABA receptor involvement in the regulation in spinal ACh release. We found that GABAA receptors are tonically inhibiting spinal ACh release. The results further suggest that GABAB receptors also are involved in the regulation of spinal ACh release. However, unlike GABAA antagonists, GABAB antagonists do not increase ACh release. This suggests that GABAB receptors are not tonically regulating the spinal ACh release.

The use of catanionic drug-surfactant mixtures was proven to be an efficient novel method of obtaining prolonged drug release from gels. It was shown that various commonly used drug compounds are able to form catanionic mixtures together with oppositely charged surfactants. These mixtures exhibited interesting phase behaviour, where, among other structures, vesicles and large worm-like or branched micelles were found. The size of these aggregates makes them a potential means of prolonging the drug release from gels, as only monomer drugs in equilibrium with larger aggregates were readily able to diffuse through the gel. When the diffusion coefficient for drug release from the formulation based upon a catanionic mixture was compared to that obtained for the drug substance and gel alone, the coefficient was some 10 to 100 times smaller. The effects of changes in the pH and ionic strength on the catanionic aggregates was also investigated, and this method of prolonging the release was found to be quite resilient to variations in both. Although the phase behaviour was somewhat affected, large micelles and vesicles were still readily found. The drug release was significantly prolonged even under physiological conditions, that is, at a pH of 7.4 and an osmolality corresponding to 0.9% NaCl. Surfactants of low irritancy, capric and lauric acid, may successfully be used instead of the more traditional surfactants, such as sodium lauryl sulfate (SDS), and prolonged release can still be obtained with ease. Some attempts to deduce the release mechanism from the proposed systems have also been made using transient current measurements, dielectric spectroscopy, and modelling of the release using the regular solution theory. In these studies, the previous assumptions made concerning the mechanism responsible for the release were confirmed to a large extent. Only small amounts of the drug existed in monomer form, and most seemed to form large catanionic aggregates with the oppositely charged surfactant.