Contaminants can exist in a wide range of states in aqueous environments, especially in surface waters. They can be freely dissolved or associated with dissolved or particulate organic matter depending on their chemical and physical characteristics. The freely dissolved fraction represents the most bioavailable fraction to an organism. These freely dissolved contaminants can cross biomembranes, potentially exerting toxic effects. Passive sampling devices (PSDs) have been developed to aid in sampling many of these contaminants by having the ability to distinguish between the freely dissolved and bound fraction of a contaminant. A new PSD, the Lipid-Free Tube (LFT) sampler was developed in response to some of the shortcomings of other current PSD that sample hydrophobic organic contaminants (HOCs). The device and laboratory methods were original modeled after a widely utilized PSD, the semipermeable membrane device (SPMD), and then improved upon. The effectiveness, efficiency, and sensitivity of not only the PSD itself, but also the laboratory methods were investigated. One requirement during LFT development was to ensure LFTs could be coupled with biological analyses without deleterious results. In an embryonic zebrafish developmental toxicity assay, embryos exposed to un-fortified LFT extracts did not show significant adverse biological response as compared to controls. Also, LFT technology lends itself to easy application in monitoring pesticides at remote sampling sites. LFTs were utilized during a series of training exchanges between Oregon State University and the Centre de Recherches en Ecotoxicologie pour le Sahel (CERES)/LOCUSTOX laboratory in Dakar, Senegal that sought to build "in country" analytical capacity. Application of LFTs as biological surrogates for predicting potential human health risk endpoints, such as those in a public health assessment was also investigated. LFT mass and accumulated contaminant masses were used directly, representing the amount of contaminants an organism would be exposed to through partitioning assuming steady state without metabolism. These exposure concentrations allow for calculating potential health risks in a human health risk model. LFT prove to be a robust tool not only for assessing bioavailable water concentrations of HOCs, but also potentially providing many insights into the toxicological significance of aquatic contaminants and mixtures. / Graduation date: 2010

6 Zusammenfassung Marianne Reinboth Bioverfügbarkeit des Flavonols Quercetin beim Hund Veterinär-Physiologisches Institut der Veterinärmedizinischen Fakultät der Universität Leipzig Eingereicht im Juni 2010 79 Seiten, 20 Abbildungen, 6 Tabellen, 211 Literaturangaben, 1 Anhang Schlüsselwörter: Quercetin, Bioverfügbarkeit, Hund, absolute Bioverfügbarkeit, Isoquercitrin, Rutin, Flavonole Für das pflanzliche Flavonol Quercetin werden vielfältige gesundheitsfördernde Wirkungen postuliert, so auch bei Hunden. Über die Bioverfügbarkeit des Flavonols bei dieser Spezies liegen bislang jedoch keinerlei Daten vor. Daher hatte diese Arbeit das Ziel, Bioverfügbarkeit und pharmakokinetische Parameter von Quercetin und wichtigen Quercetinglycosiden bei Hunden nach deren Verabreichung mit einer Testmahlzeit in einer praxisrelevanten Dosierung von 10 mg/kg Körpermasse zu untersuchen. Dazu erhielten 9 adulte Beagles beiderlei Geschlechts das zuckerfreie \"Aglycon\" Quercetin bzw. seine Glycoside Isoquercitrin (Quercetin-3-O-Glucosid) und Rutin (Quercetin-3-O-Glucorhamnosid) in jeweils äquimolarer Dosierung in einer Testmahlzeit verabreicht. Anschließend wurden Blutproben über einen Zeitraum von bis zu 72 Stunden entnommen und mittels HPLC die Konzentrations-Zeitverläufe der Metaboliten im Blutplasma, die Bioverfügbarkeit sowie weitere pharmakokinetische Parameter bestimmt. Weiterhin wurde die absolute Bioverfügbarkeit von Quercetin aus dem Vergleich einer oralen mit einer intravenösen Applikation bestimmt. Der weitaus größte Teil der Plasmametaboliten von Quercetin sowie seiner beiden Glycoside bestand aus glucuronidierten bzw. sulfatierten Quercetinkonjugaten. Nicht konjugiertes Quercetin-Aglycon kam nur in einem Anteil von etwa 20 % vor. Neben Quercetin machten seine Metaboliten Isorhamnetin und Kämpferol weniger als 10 % aller im Plasma zirkulierenden Flavonole aus. Die absolute Bioverfügbarkeit von Quercetin betrug nur etwa 4 %. Die relative Bioverfügbarkeit aus dem 3-O-Glucosid Isoquercitrin war mehr als doppelt so hoch wie aus dem Aglycon, die maximalen Plasmaspiegel lagen aber auch hier unter 1 µmol/l. Sowohl nach Aufnahme von Quercetin als auch nach Isoquercitrin kam es zu einer relativ schnellen Absorption aus dem Dünndarm mit einem ersten Plasmapeak ungefähr eine Stunde nach der Ingestion. Vier Stunden nach Aufnahme der beiden Flavonole trat ein zweiter Plasmapeak auf, der in der Regel höher als der erste ausfiel. Dies deutet auf einen enterohepatischen Kreislauf der über die Galle ausgeschiedenen Metaboliten hin. Nach Aufnahme von Rutin kam es zu einer verzögerten Absorption, da eine Deglycosylierung durch bakterielle Glycosidasen im Dickdarm Voraussetzung für die Absorption des Flavonols ist. Maximale Plasmakonzentrationen wurden im Mittel erst 11 Stunden nach Ingestion dieses Glycosids erreicht. Die maximalen Plasmakonzentra-tionen nach Rutin waren geringer als nach Quercetin oder Isoquercitrin, jedoch war die mittlere Verweildauer der Plasmametaboliten mit 18 Stunden auch wesentlich länger. Im Unterschied zu anderen Spezies war die relative Bioverfügbarkeit von Rutin gegenüber Quercetin nicht verringert. Obwohl Rutin eine relativ gute Quercetinquelle für Hunde zu sein scheint, muss bei der Einschätzung möglicher In-vivo-Wirkungen die relativ geringe Bioverfügbarkeit sowie die intensive Metabolisierung seines Aglycons Quercetin berücksichtigt werden.:1 Einleitung 1 2 Literaturübersicht 3 2.1 Funktionen von Flavonoiden in Pflanzen 3 2.2 Biosynthese und Struktur von Flavonoiden 4 2.3 Flavonoidwirkungen im menschlichen und tierischen Organismus 7 2.3.1 Antioxidative Eigenschaften 10 2.3.2 Wirkungen auf Enzyme und Transportproteine des Arzneistoffwechsels 12 2.4 Bioverfügbarkeit und Stoffwechsel des Flavonols Quercetin 13 2.4.1 Bioverfügbarkeit von Quercetin 13 2.4.2 Einfluss des Futters 15 2.4.3 Einfluss des Glycosylierungsmusters 16 2.4.4 Intestinale Absorption und Metabolismus 18 2.4.5 Einfluss der intestinalen Mikroflora 21 2.4.6 Bindung an Plasmaproteine 22 2.4.7 Gewebeverteilung 23 2.4.8 Exkretion 24 2.5 Zielsetzung 25 3 Tiere, Material und Methoden 26 3.1 Versuchstiere und Haltungsbedingungen 26 3.2 Verwendete Flavonole 27 3.3 Durchführung des Tierversuchs 28 3.3.1 Allgemeine Durchführung der Versuche 28 3.3.2 Bioverfügbarkeit von Quercetin und Rutin (je 30 mg/kg KM) 29 3.3.3 Absolute Bioverfügbarkeit von Quercetin 30 3.3.4 Relative Bioverfügbarkeit verschiedener Quercetinglycoside 31 3.4 Probenaufarbeitung 31 3.5 HPLC 33 3.5.1 Methodenvalidierung und -kalibrierung 34 3.5.2 Berechnung der pharmakokinetischen Parameter 40 3.5.3 Statistische Auswertung 41 4 Ergebnisse 42 4.1 Bioverfügbarkeit von Quercetin und Rutin (je 30 mg/kg KM) 42 4.1.1 Quercetin-Aglycon 42 4.1.2 Rutin 46 4.2 Absolute Bioverfügbarkeit Quercetin 48 4.3 Relative Bioverfügbarkeit verschiedener Quercetinglycoside 52 4.3.1 Quercetin-Aglycon 53 4.3.2 Rutin 56 4.3.3 Isoquercitrin 57 5 Diskussion 62 5.1 Zielsetzung der Studie 62 5.2 Methodische Aspekte 62 5.2.1 Auswahl der Versuchstiere und Versuchsanordnung 62 5.2.2 Wahl der Testmahlzeit 63 5.2.3 Einfluss der Zeitpunkte für die Probennahme auf die Berechnung der Verfügbarkeit 64 5.2.4 Analysemethode 65 5.3 Plasmametaboliten von Quercetin 66 5.4 Absolute Bioverfügbarkeit von Quercetin 69 5.5 Relative Bioverfügbarkeit verschiedener Quercetinglycoside 70 5.5.1 Isoquercitrin 70 5.5.2 Rutin 71 5.6 Bezug der pharmakokinetischen Daten zu potentiellen In-vivo-Wirkungen 73 5.7 Schlussfolgerungen 75 6 Zusammenfassung 76 7 Summary 78 8 Literaturverzeichnis 80 9 Anhang 102 9.1 HPLC-Chemikaien 102 9.2 Validierung der HPLC-Methode 103 Danksagung 109 / 7 Summary Marianne Reinboth Bioavailability of the Flavonol Quercetin in Dogs Institute of Physiology of the Faculty of Veterinary Medicine, University of Leipzig Submitted in June 2010 79 pages, 20 figures, 6 tables, 211 references, 1 appendix Keywords: quercetin, bioavailability, dog, absolute bioavailability, isoquercitrin, rutin, flavonols The plant flavonol quercetin is supposed to exert multiple health-related effects in dogs. To date no information on its bioavailability in this particular species is avai-lable. This study intended to investigate bioavailability and pharmacokinetics of quercetin and certain quercetin glycosides in dogs after ingestion of a test meal sup-plemented with a quercetin dose equivalent to 10 mg/kg body weight. Nine adult beagle dogs of both sexes received the aglycon quercetin (sugarfree) or its glycosides isoquercitrin (quercetin-3-O-glucoside) and rutin (quercetin-3-O-glucorhamnoside) in equimolar amounts together with a test meal. Blood samples were taken over a period of up to 72 hours; bioavailability and pharmacokinetics were calculated from the HPLC-derived plasmaconcentration-time-curves. Absolute bioavailability was calculated by comparing an oral to an intravenous administration of quercetin. The majority of analysed plasma metabolites were glucuronidated and sulfated con-jugates of quercetin. Non-conjugated quercetin aglycon comprised only 20 %. Be-sides quercetin, its metabolites isorhamnetin and kaempferol made up less than 10 % of all circulating metabolites. The absolute bioavailability of quercetin was only 4 %. The relative bioavailability of quercetin from isoquercitrin was more than twice as high than from the aglycon, but even there maximal plasma concentrations were generally less than 1 μmol/l. Absorption from the small intestine was rather fast with a first plasma peak after 1 hour after ingestion of quercetin or isoquercitrin. A second, generally higher plasma peak occurred 4 hours after ingestion. This suggests an in-tensive enterohepatic recycling of biliary secreted metabolites. Absorption was significantly delayed after ingestion of rutin due to the necessity of bacterial deglycosilation in the large intestine. Plasma concentrations peaked only after 11 hours. Plasma concentrations after rutin were lower than after quercetin or isoquercitrin, but mean residence time of plasma metabolites was as long as 18 hours after rutin ingestion. Consequently, a once daily feeding of dogs with rutin might lead to relatively constant plasma metabolite concentrations. In contrast to other species, bioavailability from rutin was not smaller than that from quercetin. Although rutin seems to be a relative good quercetin source for dogs, estimations about potential in-vivo-effects of quercetin have to take into consideration its low bioavailabilty and intensive metabolism.:1 Einleitung 1 2 Literaturübersicht 3 2.1 Funktionen von Flavonoiden in Pflanzen 3 2.2 Biosynthese und Struktur von Flavonoiden 4 2.3 Flavonoidwirkungen im menschlichen und tierischen Organismus 7 2.3.1 Antioxidative Eigenschaften 10 2.3.2 Wirkungen auf Enzyme und Transportproteine des Arzneistoffwechsels 12 2.4 Bioverfügbarkeit und Stoffwechsel des Flavonols Quercetin 13 2.4.1 Bioverfügbarkeit von Quercetin 13 2.4.2 Einfluss des Futters 15 2.4.3 Einfluss des Glycosylierungsmusters 16 2.4.4 Intestinale Absorption und Metabolismus 18 2.4.5 Einfluss der intestinalen Mikroflora 21 2.4.6 Bindung an Plasmaproteine 22 2.4.7 Gewebeverteilung 23 2.4.8 Exkretion 24 2.5 Zielsetzung 25 3 Tiere, Material und Methoden 26 3.1 Versuchstiere und Haltungsbedingungen 26 3.2 Verwendete Flavonole 27 3.3 Durchführung des Tierversuchs 28 3.3.1 Allgemeine Durchführung der Versuche 28 3.3.2 Bioverfügbarkeit von Quercetin und Rutin (je 30 mg/kg KM) 29 3.3.3 Absolute Bioverfügbarkeit von Quercetin 30 3.3.4 Relative Bioverfügbarkeit verschiedener Quercetinglycoside 31 3.4 Probenaufarbeitung 31 3.5 HPLC 33 3.5.1 Methodenvalidierung und -kalibrierung 34 3.5.2 Berechnung der pharmakokinetischen Parameter 40 3.5.3 Statistische Auswertung 41 4 Ergebnisse 42 4.1 Bioverfügbarkeit von Quercetin und Rutin (je 30 mg/kg KM) 42 4.1.1 Quercetin-Aglycon 42 4.1.2 Rutin 46 4.2 Absolute Bioverfügbarkeit Quercetin 48 4.3 Relative Bioverfügbarkeit verschiedener Quercetinglycoside 52 4.3.1 Quercetin-Aglycon 53 4.3.2 Rutin 56 4.3.3 Isoquercitrin 57 5 Diskussion 62 5.1 Zielsetzung der Studie 62 5.2 Methodische Aspekte 62 5.2.1 Auswahl der Versuchstiere und Versuchsanordnung 62 5.2.2 Wahl der Testmahlzeit 63 5.2.3 Einfluss der Zeitpunkte für die Probennahme auf die Berechnung der Verfügbarkeit 64 5.2.4 Analysemethode 65 5.3 Plasmametaboliten von Quercetin 66 5.4 Absolute Bioverfügbarkeit von Quercetin 69 5.5 Relative Bioverfügbarkeit verschiedener Quercetinglycoside 70 5.5.1 Isoquercitrin 70 5.5.2 Rutin 71 5.6 Bezug der pharmakokinetischen Daten zu potentiellen In-vivo-Wirkungen 73 5.7 Schlussfolgerungen 75 6 Zusammenfassung 76 7 Summary 78 8 Literaturverzeichnis 80 9 Anhang 102 9.1 HPLC-Chemikaien 102 9.2 Validierung der HPLC-Methode 103 Danksagung 109

Doctor of Philosophy / Department of Agronomy / Ganga M. Hettiarachchi / Urban gardens have been a popular re-use option in the transformation of brownfields—located in older industrialized cities and near peri-urban developments. They provide accessible, available, and affordable supplies of fresh fruits and vegetables, effectively reducing the enigma of “food deserts” across U.S. cities. However, direct (soil ingestion, inhalation) and indirect (soil-plant-human) human exposure concerns about real or perceived trace element contamination in urban soils persist due to previous use. Elevated lead (Pb) and/or arsenic (As) concentrations were found at two (Tacoma and Seattle, WA) urban gardens. The Tacoma site was contaminated with Pb (51 to 312 mg kg-1) and As (39 to 146 mg kg-1), whereas soil Pb at the Seattle site ranged from 506 to 2,022 mg kg-1, and As concentrations were < 20 mg kg-1. Experimental design at both sites was a randomized complete block with a split-plot arrangement (main plots: biosolids/compost vs. non-amended control; sub-plot: plant type). Tacoma site treatment included a Class A biosolids mix (TAGRO) with dolomite. The Seattle site was amended with Cedar-Grove Compost (CGC) plus dolomite. Efficacy of biosolids/compost amendment in reducing Pb and As concentrations was evaluated using root, leafy, and fruit vegetables. Soil Pb and As bioaccessibility were also evaluated. Food chain transfer of Pb and As in vegetables due to surface contamination of produce samples were evaluated on the basis of cleaning procedures. A laboratory incubation study and a controlled greenhouse experiment were conducted on soils collected from the Tacoma site. Effectiveness of addition of laboratory synthesized ferrihydrite (Fh: iron oxyhydroxide) and TAGRO mix, each alone or in combination were screened and tested on the Pb and As co-contaminated Tacoma soil. Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy studies of Pb and As were conducted on incubation study samples to understand treatment-induced Pb- and As-speciation changes. Dilution of soil Pb (10 to 23%) and As (12 to 25%) were observed for biosolids amendment at the Tacoma site, while CGC amendment resulted in 20 to 50% dilution in soil Pb at the Seattle site. Biosolids and CGC amendments reduced Pb concentrations in the vegetables by 50% to 71%. At both sites, Pb concentrations of root vegetables exceeded the MLs established by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO). Arsenic concentrations in vegetables were below an estimated ML and were reduced by 46% to 80% when grown on biosolids amended soils. Laboratory cleaning further reduced Pb and As food-chain transfer in vegetables grown in contaminated urban soils. Laboratory incubation and greenhouse studies showed dissolution of Pb in TAGRO plus Fh, and Pb concentrations in Fh amendments were significantly lower than the other amendments. Bioaccessible Pb and As were low. Significant reductions in bioaccessible As were observed when soils were amended with both TAGRO and Fh. X-ray absorption spectroscopy results indicated that chloropyromorphite-like (stable Pb phosphates) phases were the most dominant Pb species. Arsenic existed mainly as As5+, scorodite (FeAsO4•2H2O)-like species in all the treatments ranging from about 60% (control) to about 70% (TAGRO plus ferrihydrite). Amendments utilizing both biosolids and Fh significantly reduce human exposure risks present in urban soils contaminated with Pb and As.

Eye disorders are on the rise as a result of an ageing population, an increasing obesity problem and a growth in the number of diabetic patients. Conventional ophthalmic formulations do not maintain therapeutic drug concentration in the target tissues for a long duration due to the physiological and anatomical eye barriers. Novel delivery systems such as nanoparticles have been explored to enhance the delivery of therapeutic agents to the eye. These delivery systems have in general been assessed using in-vivo animal models, despite ethical concerns for animal wellbeing. The aims of this thesis were to synthesise and characterise four amphiphilic polymers, subsequently prepare and characterise four nano sized polymeric self-assemblies loaded with triamcinolone acetonide (TA), develop an in-vitro porcine eye model and to evaluate the permeation of nano sized self-assemblies using the developed model. Four comb-shaped amphiphilic polymers (Pa5, Pa5-MPEG, Ch5 and Da10) were synthesised with a high yield (>81%) and good reproducibility. These polymers formed spontaneous positive self-assemblies in aqueous media (114-314 nm). The mean hydrodynamic diameters of the positive spontaneous self-assemblies entrapping TA were in the range of 200–334 nm loading high concentrations (455-1263 μg mL-1) of TA, much greater than the TA inherent aqueous solubility or concentrations achieved using conventional solubilisers. A porcine in-vitro eye model was developed to assess drug permeation through anterior and posterior ocular tissues. The model was partially validated using tritiated water and a series of hydrophilic markers with increasing molecular weights. The integrity of porcine ocular tissue was checked by monitoring the permeation of tritiated water to ensure the membrane intactness. Tritiated water permeation at 15 min was exploited as a potential method to normalise drug flux, as tritiated water percentage permeation at 15 min had an inverse relationship with tissue thickness (R2 = 0.66), to reduce the inherent variability between tissue samples thus increasing the accuracy of the in-vitro eye model. Four markers (fluorescein sodium salt, 4, 10 and 20 kDa FITC-dextran) were used for the purpose of investigating the effect of increasing molecular weight on ocular tissue permeability. The permeability of the markers displayed an inverse relationship and abrupt decline with Mw in terms of the permeability through scleral and corneal tissues of human and porcine and the molecular weight of the markers. The developed porcine in-vitro eye model showed good correlation with the human in-vitro model providing strong evidence it can be used to screen potential formulations before testing in-vivo. The TA loaded self-assemblies and a few chemical enhancers (glutamic acid, tween 80, chitosan, Pa5 and elevate temperature (45 °C)), selected to assist drug delivery via two routes (paracellular and transcellular), were tested using the developed in-vitro eye model. The results showed there was no marker permeation enhancement effect in porcine and human ocular tissues using chemical enhancers. In summary, a porcine in-vitro eye model was developed to assess hydrophobic and hydrophilic penetrant permeation across anterior and posterior ocular tissues. The porcine in-vitro eye model showed good correlation with the human in-vitro model providing strong evidence that the porcine in-vitro eye model can be used to screen potential formulations before testing in-vivo using the porcine model which ultimately might correlate well with the in-vivo human responses. Although TA self-assemblies did not significantly increase drug flux through human or porcine scleral tissues, it might be of interest for ophthalmic topically administered formulations due to their positive charge and small nano size.

The primary aim was to validate and implement a urinary pharmacokinetic method for terbutaline to determine the relative lung and systemic bioavailability following inhalation and to measure the in-vitro characteristics of the emitted dose by these inhalation methods. Two new robust, accurate and sensitive high performance liquid chromatography methods for the determination of terbutaline in aqueous and urine samples were validated in accordance with the FDA and ICH guidelines. Terbutaline was extracted using solid phase extraction with salbutamol and bamethane as internal standards. The accuracy, precision, lower limit of detection and recovery for both methods were within recognized limits. The in-vitro characteristics of terbutaline sulphate inhalers were measured according to standard compendial methodology as well as adaptation of this methodology to simulate routine patient use. The dose emission of terbutaline sulphate from a Bricanyl Turbuhaler was determined using an inhalation volume of 4 L at inhalation flows of 10-60 L min-1. The particle size distribution was measured using an Anderson Cascade Impactor (ACI) with a mixing inlet valve to allow measurement at different flows. A steady increase in total emitted dose (TED) and the fine particle dose (FPD) was observed as the inhalation flow increased thereby highlighting the flow dependent dose emission characteristics of the Turbuhaler. The in-vitro dose emission characteristics of terbutaline sulphate from Bricanyl MDIs were measured according to the standard compendial methodology at a flow of 28.3 L min-1 using a 4 L inhalation volume. The TED and particle size distribution of terbutaline sulphate from the Bricanyl MDI were determined alone and with different spacers [AeroChamber Max (AMAX), AeroChamber Plus (APLUS), Fisonair and Nebuhaler]. The TED from the MDI alone was significantly higher than all MDI+spacers (p<0.001). The MDI with APLUS resulted in the smallest mass median aerodynamic diameter (MMAD) and the highest fine particle fraction (FPF). The MDI with AMAX resulted in the highest FPD. The in-vitro characteristics of terbutaline sulphate from Bricanyl respules using the Aeroneb Pro (vibrating mesh) and Sidestream jet nebulisers were determined by the CEN methodology and the Next Generation Impactor (NGI) methodology. The Aeroneb Pro was found to have significantly better aerodynamic properties than the Sidestream. The results from the NGI method were significantly different from the CEN method suggesting further evaluation of both methods. Cooling the NGI decreased the evaporation effect. Twelve healthy volunteers (6 females) completed in-vivo urinary terbutaline pharmacokinetic studies to determine the relative bioavailability following inhalation. The differences between the amounts excreted 0.5, 1, 2, 4, 6 and 24 hour post inhalation from a Bricanyl MDI (I) and oral (O) dosing of 500 µg terbutaline sulphate and with the co-administration of oral charcoal (IC and OC, respectively) were studied. No terbutaline was found in OC samples. The amount of terbutaline excreted 30 minutes post I and IC were significantly (p<0.001) higher than post O suggesting that the amount of terbutaline excreted 30 minutes post dosing can be used as an index of the lung deposition. The amount of terbutaline excreted 24 hour post I was significantly (p<0.01) higher than post O suggesting that the amount of terbutaline excreted 24 hour post dosing can be used as an index of the relative systemic bioavailability. The dose response relationships and the low inter and intra-subject variability studies confirm the feasibility of this method. To demonstrate the application of the method the effect of inhalation technique on the lung and systemic bioavailability following inhalation from a dry powder inhaler was evaluated. The effect of different spacers on the dose emitted from the Bricanyl MDI and the effect of different nebulisers on the dose emitted were also studied using twelve healthy volunteers (6 females) for each study. A fast inhalation flow using the Bricanyl Turbuhaler resulted in significantly higher amounts of terbutaline excreted 0.5 and 24 hour post dosing (2 doses of 500µg terbutaline sulphate from Bricanyl Turbuhaler) than slow inhalation flow (p<0.001). The Bricanyl MDI alone resulted in a significantly higher amount of terbutaline excreted 24 hour post dosing (2 doses of 250µg terbutaline sulphate from Bricanyl MDI) and significantly lower amounts excreted 30 minutes post dosing than the MDI+Spacers. The AMAX provided a greater amount of urinary terbutaline excreted 30 minutes post dosing than the APLUS and Nebuhaler. The Aeroneb Pro resulted in significantly higher amounts of terbutaline excreted 0.5 and 24 hour post dosing (1 dose of 5mg/2ml terbutaline sulphate from Bricanyl respule) than a Sidestream Jet nebuliser (p<0.001). Further application of the method was demonstrated by 12 (6 female) COPD non-invasive mechanically ventilated patients. One dose of 2mg in 0.8ml terbutaline sulphate respiratory solution from Aeroneb Pro and one dose of 5mg in 2ml terbutaline sulphate respiratory solution from Sidestream jet nebuliser resulted in a similar amounts of urinary terbutaline excreted 0.5 and 24 hour post dosing. The results were consistent with the results of the ex-vivo study performed on the same patients. The thesis highlights extension of the urinary pharmacokinetic method following inhalation to terbutaline and its application in volunteer and patient studies.

Over recent years the delivery of nanosized drug particles has shown potential in improving bioavailability. Drug nanosizing is achieved by 'top-down' and by 'bottom-up' approaches. Owing to limitations associated with the top-down techniques, such as high energy input, electrostatic effects, broad particle size distributions and contamination issues, great interest has been directed to alternative bottom up technologies. In this study, the hypothesis that microreactors can be used as a simple and cost-effective technique to generate organic nanosized products is tested using three steroids (hydrocortisone, prednisolone and budesonide). Arrested antisolvent nanoprecipitation using ethanol (solvent) and water (antisolvent) was conducted within the microreactors. To enable experimental design for the microreactor studies, solubility profiles in different ethanol-water combinations at 25 °C were explored. All three drugs' solubility increased with increasing ethanol concentration showing maxima at 80-90 % v/v ethanol-water mixtures. Because of the complex multivariate microfluidic process, artificial neural network modelling was then employed to identify the dominant relationships between the variables affecting nanoprecipitation (as inputs) and the drug particle size (as output). The antisolvent flow rate was found to have the major role in directing drug particle size. Based on these successful findings, the potential of preparing pharmaceutical nanosuspensions using microfluidic reactors was researched. A hydrocortisone (HC) nanosuspension (NS) was prepared by introducing the generated drug particles into an aqueous solution of stabilizers stirred at high speed with a propeller mixer. A tangential flow filtration system was then used to concentrate the prepared NS. Results showed that a stable narrow sized HC NS of amorphous spherical particles 500 ± 64 nm diameter and zeta potential -18 ± 2.84 mV could be produced. The ocular bioavailability of a microfluidic precipitated HC NS (300 nm) was assessed and compared to a similar sized, milled HC NS and HC solution as a control. The precipitated and the milled NS achieved comparable AUC0-9h of 28.06 ± 4.08 and 30.95 ± 2.2, respectively, significantly (P < 0.01) higher than HC solution (15.86 ± 2.7). These results illustrate the opportunity to design sustained release ophthalmic formulations. Going nano via microfluidic precipitation was also exploited to tailor budesonide (BD) NS for pulmonary administration. The in vitro aerosolization by nebulization of a BD NS was studied in comparison with a commercial BD microsuspension. Overall, the fine particle fraction generated from BD NS (56.88 ± 3.37) was significantly (P < 0.05) higher than the marketed BD (38.04 ± 7.81). The mean mass aerodynamic diameter of BD NS aerosol (3.9 ± 0.48 μm) was significantly smaller (P < 0.05) than the microsuspension (6.2 ± 1.09 μm) indicating improved performance for BD NS. In conclusion, findings of this study support the hypothesis of using microfluidic nanoprecipitation as a promising and economical technique of drug nanosizing.

The purpose of this project was to fabricate biodegradable ophthalmic inserts for controlled delivery of pilocarpine and evaluate them by both in-vitro and in-vivo studies. Emphasis was placed on the use of an inexpensive material as a drug carrier and on the ease of fabrication of the device. Based on these criteria, absorbable gelatin was selected to fabricate a matrix system. Absorbable gelatin can be obtained by either thermal treatment or chemical crosslinking of gelatin. In the first part of this project, we fabricated an insert using Gelfoamᴿ, an absorbable gelatin sponge obtained by thermal treatment. A prolonged in-vitro release of pilocarpine from the device was achieved through pharmaceutical modification by embedding a retardant in the pores. The devices impregnated with polyethylene glycol monostearate (PMS) and cetyl esters wax (CEW) were found to be most effective. The in-vivo evaluation of the devices indicated that pharmaceutical modification of Gelfoamᴿ is an effective means of improving the biological activity of pilocarpine without altering the biodegradability of the biopolymer backbone. The CEW device produces a substantial improvement in drug bioavailability and an increase in the duration of biological effect over that from the two commercial formulations, the eyedrop and the gel. In the second part of the project, we fabricated absorbable gelatin inserts through chemical crosslinking of gelatin. The effect of selected fabrication variables on profiles of the in-vitro release of pilocarpine and the dynamic water uptake by the crosslinked gelatin devices was investigated. These results were further substantiated by the measurement of the degree of crosslinking of gelatin. The in-vivo study indicated that the modification of the structure of gelatin by crosslinking is another simple and effective way of improving bioavailability and extending the duration of effect of pilocarpine incorporated in the biopolymeric device. In addition, altering the degree of crosslinking of gelatin allows a variation of the biodegradation time of the polymer.

Norint ištirti vario (Cu) ir kadmio (Cd) įtaką augalams, augintiems skirtingose terpėse, buvo vertinamas diferencijuotas ir kompleksinis sunkiųjų metalų poveikis jų morfologiniams ir fiziologiniams rodikliams. Varis reikalingas mažais kiekiais kaip mitybos elementas, bet tampa toksišku esant didesnėms jo koncentracijoms aplinkoje. Kadmis neturi biologinės funkcijos ir gali būti toksiškas esant mažoms koncentracijoms, priklausomai nuo aplinkos sąlygų, jo cheminio būvio ir t.t. Tyrimo objektais buvo pasirinkti vasariniai miežiai (Hordeum vulgare L.) ir mažosios plūdenos (Lemna minor). Šio darbo tikslas yra įvertinti ir palyginti Cd ir Cu poveikį esant skirtingam biologiniam prieinamumui iš vandeninės terpės ir dirvožemio. Tiriamas darbas buvo atliekamas dviem etapais. Visų pirma buvo tiriamas pavienis metalų poveikis Lemna minor. Savaitę jos buvo auginamos 0,1 µM; 0,5 µM; 1 µM; 10 µM; 50 µM ir 100 µM koncentracijos kadmio bei 1 µM; 5 µM; 10 µM; 50 µM ir 100 µM koncentracijos vario tirpaluose. Metalų mišinių koncentracijos, kuriomis buvo veikiamos Lemna minor, pasirinktos pagal diferencituotas, kuriose augalai išgyveno daugiau kaip pusę tiriamojo laikotarpio. Nustačius EC50 koncentracijas pagal tiriamojo parametro, lyginant su kontrole, sumažėjimą, apskaičiuota EC50 mišinio. Mišinio koncentracijos išreikštos kaip TU. Įvertinti Lemna minor biomasės prieaugio, augimo inhibicijos pokyčiai. Kompleksinio poveikio atveju augimo greičiui (EC50 mišinio = 2,5 TU) ir biomasės... [toliau žr. visą tekstą] / The aim of this study was to investigate the influence of cadmium (Cd) and copper (Cu) to plants froms diferent growing medium and evaluate the single and combined effect to their morphologic and physiologic parameters. Cu is known as micronutrient. It is necessary for plants but becames toxic at higher concentrations. Cd doesn‘t have any biological function and can be toxic even at small concentrations. It‘s toxicity depends on environmental conditions, chemical state and etc. The subject of this research is agricultural plants summer barley (Hordeum vulgare L.) and water plants duckweed (Lemna minor). The purpose of study is to value and to compare Cd and Cu effects to plants from water and soil medium. The research was done in two stages. At first, the single heavy metal effect to Lemna minor was investigated. Plants were treated one week by 0,1 µM; 0,5 µM; 1 µM; 10 µM; 50 µM; 100 µM Cd and 1 µM; 5 µM; 10 µM; 50 µM; 100 µM Cu concentrations. Considered to single concentrations, combained concentrations were chosen. Concentrations in the mixtures was expressed as TU, as fraction of their median affective concentration (EC50). Biomass and growing inhibition were measured. Antagonistic responses to biomass (EC50 mix = 2 TU) and growth inhibition (EC50 mix = 2,5 TU) occurred with Cd+Cu mixture. The experiment was also done with summer barley (Hordeum vulgare L.). Two weeks they were grown in soil and water solutions affected by the same heavy metal concentrations like Lemna... [to full text]

In today’s drug discovery, the number of candidate drugs based on new molecular entities with poor aqueous solubility is increasing. Since poor aqueous solubility of an active pharmaceutical ingredients (APIs) is associated with low bioavailability and thus limite their therapeutic effect, this is often a great challenge in the development of new drugs when oral administration is the preferred route of administration. A number of different strategies have been developed to circumvent this problem where salt formulations of an API is the most widely employed method. However, new strategies are needed since there is no one solution that solves this issue for all substances. In recent time, the concept of stabilizing poorly soluble APIs in their amorphous form has gained a lot of attention since amorphous compounds exhibit a higher apparent solubility compared to their crystalline counterparts. Amorphous substances are prone to crystallize if left in a non-constricted environment and thus need to be stabilized if the amorphous state is to be conserved until administration. Inorganic mesoporous materials have been proposed as an interesting type of excipients that can conserve the amorphous state of APIs. In this work, the focus was to investigate the possibilities of using a mesoporous type of magnesium carbonate to stabilize the amorphous state of different APIs. Due to the nanometer sized pores in the material, complete conservation of amorphous APIs was obtained. This resulted in both an increase in in vitro release rate and a higher solubility of the substances which may translate to both a faster onset of action and an improved therapeutic effect of the APIs in a clinical situation. The long term stability of formulations was also investigated showing promising results. The results presented in this work show that mesoporous magnesium carbonate represents an interesting type of excipient for oral formulations of APIs with poor aqueous solubility. / <p>Felaktigt ISBN 978-91-554-9702-6 i tryck version.</p>

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