Development of a novel in vitro system for nasal drug delivery development

There is currently, no ideal system for studying nasal drug delivery in vitro. The existing techniques such as the Ussing chamber and cell culture all have major disadvantages. Most importantly, none of the existing techniques accurately represent the interior of the nasal cavity, with its airflow and humidity; neither do they allow the investigation of solid dosage forms.The work in this thesis represents the development of an in vitro model system in which the interior characteristics of the nasal cavity are closely represented, and solid or minimal volume dosage forms can be investigated. The complete nasal chamber consists of two sections: a lower tissue, viability chamber and an upper nasal chamber. The lower tissue viability chamber has been shown, using existing tissue viability monitoring techniques, to maintain the viability of a number of epithelial tissues, including porcine and rabbit nasal tissue, and rat ileal and Payers' patch tissue. The complete chamber including the upper nasal chamber has been shown to provide tissue viability for porcine and rabbit nasal tissue above that available using the existing Ussing chamber techniques. Adaptation of the complete system, and the development of the necessary experimental protocols that allow aerosol particle-sizing, together with videography, has shown that the new factors investigated, humidity and airflow, have a measurable effect on the delivered dose from a typical nasal pump. Similarly, adaptation of the chamber to fit under a confocal microscope, and the development of the necessary protocols has shown the effect of surface and size on the penetration of microparticulate materials into nasal epithelial tissues. The system developed in this thesis has been shown to be flexible, in allowing the development of the confocal and particle-sizing systems. For future nasal drug delivery studies, the ability to measure such factors as the size of the delivered system in the nasal cavity, the depth of penetration of the formulation into the tissue are essential. Additionally, to have access to other data such as that obtained from drug transport in the same system, and to have the tissue available for histological examination represents a significant advance in the usefulness of such an in vitro technique for nasal delivery.

The development of a novel in vitro model suitable for the prediction of bioavailability

In vitro studies of drug absorption processes are undertaken to assess drug candidate or formulation suitability, mechanism investigation, and ultimately for the development of predictive models. This study included each of these approaches, with the aim of developing novel in vitro methods for inclusion in a drug absorption model. Two model analgesic drugs, ibuprofen and paracetamol, were selected. The study focused on three main areas, the interaction of the model drugs with co-administered antacids, the elucidation of the mechanisms responsible for the increased absorption rate observed in a novel paracetamol formulation and the development of novel ibuprofen tablet formulations containing alkalising excipients as dissolution promoters.Several novel dissolution methods were developed. A method to study the interaction of drug/excipient mixtures in the powder form was successfully used to select suitable dissolution enhancing exicipents. A method to study intrinsic dissolution rate using paddle apparatus was developed and used to study dissolution mechanisms. Methods to simulate stomach and intestine environments in terms of media composition and volume and drug/antacid doses were developed. Antacid addition greatly increased the dissolution of ibuprofen in the stomach model.Novel methods to measure drug permeability through rat stomach and intestine were developed, using sac methodology. The methods allowed direct comparison of the apparent permeability values obtained. Tissue stability, reproducibility and integrity was observed, with selectivity between paracellular and transcellular markers and hydrophilic and lipophilic compounds within an homologous series of beta-blockers.

Biological barriers to the nasal delivery of peptide drugs

The nasal absorption of larger peptide and protein drugs is generally low. The importance of the mucus layer and enzymic degradation in reducing absorption were investigated. Reversed-phase high-performance liquid chromatographic (HPLC) methods were developed to assay a variety of compounds. Pig gastric mucus (PGM) was selected to investigate the importance of the mucus layer. A method of treating and storing PGM was developed and evaluated which was representative of the gel in vivo. The nature of the mucus barrier was evaluated in vitro with three-compartment diffusion cells and a series of compounds with differing physicochemical properties. Mucus retarded the diffusion of all the compounds with molecular weight and charge exerting a marked effect. Binding to mucus was investigated by a centrifugation method. All of the compounds tested were found to bind to mucus with the exception of the negatively charged molecule benzoic acid. The small peptides did not demonstrate greater binding to mucus than any of the other compounds evaluated. The effect of some absorption enhancers upon the rate of diffusion of tryptophan through mucus was determined in vi tro. At the concentrations employed the enhancers EDTA, N-acetylcysteine and taurodeoxycholic acid exerted no effect, whilst taurocholic acid and cholic acid, were found to slightly reduce the rate of diffusion. The intracellular and luminal proteolytic activity of the nose was investigated in the sheep animal model with a nasal mucosal homogenate and a nasal wash preparation respectively and a series of chemically similar peptides. Hydrolysis was also investigated with the proteolytic enzymes carboxypeptidase A, cytosolic leucine aminopeptidase and microsomal leucine aminopeptidase. Sheep nasal mucosa possesses significant peptide hydrolase activity capable of degrading all the substrates tested. Considerable variation in susceptibility was observed. Degradation occurred excl us i ve ly at the pept ide bond between the aromatic amino ac id and glycine, indicating some specificity for aromatic amino acids. Hydrolysis profiles indicated the presence of both aminopeptidase and carboxypeptidase enzymes. The specific activity of the microsomal fraction was found to be greater than the cytosolic fraction. Hydrolysis in the nasal wash indicated the presence of either luminal or loosely-bound proteases, which can degrade peptide substrates. The same specificity for aromatic amino acids was observed and aminopeptidase activity demonstrated. The specific activity of the nasal wash was smaller than that of the homogenate.

The role of H2S bioavailability in endothelial dysfunction

Endothelial dysfunction (EDF) reflects pathophysiologicalchanges in the phenotype and functions of endothelial cells that result fromand/or contribute to a plethora of cardiovascular diseases. We review the roleof hydrogen sulfide (H2S) in the pathogenesis of EDF, one of thefastest advancing research topics. Conventionally treated as an environmentpollutant, H2S is also produced in endothelial cells and participatesin the fine regulation of endothelial integrity and functions. Disturbed H2Sbioavailability has been suggested to be a novel indicator of EDF progress andprognosis. EDF manifests in different forms in multiple pathologies, buttherapeutics aimed at remedying altered H2S bioavailability maybenefit all.

Stream dissolved organic matter bioavailability and composition in watersheds underlain with discontinuous permafrost

We examined the impact of permafrost on dissolved organic matter (DOM) composition in Caribou-Poker Creeks Research Watershed (CPCRW), a watershed underlain with discontinuous permafrost, in interior Alaska. We analyzed long term data from watersheds underlain with varying degrees of permafrost, sampled springs and thermokarsts, used fluorescence spectroscopy, and measured the bioavailabity of dissolved organic carbon (DOC). Permafrost driven patterns in hydrology and vegetation influenced DOM patterns in streams, with the stream draining the high permafrost watershed having higher DOC and dissolved organic nitrogen (DON) concentrations, higher DOC:- DON and greater specific ultraviolet absorbance (SUVA) than the streams draining the low and medium permafrost watersheds. Streams, springs and thermokarsts exhibited a wide range of DOC and DON concentrations (1.5–37.5 mgC/L and 0.14–1.26 mgN/L, respectively), DOC:DON (7.1–42.8) and SUVA (1.5–4.7 L mgC-1 m-1). All sites had a high proportion of humic components, a low proportion of protein components, and a low fluorescence index value (1.3–1.4), generally consistent with terrestrially derivedDOM. Principal component analysis revealed distinct groups in our fluorescence data determined by diagenetic processing and DOM source. The proportion of bioavailable DOC ranged from 2 to 35%, with the proportion of tyrosine- and tryptophan-like fluorophores in the DOM being a major predictor of DOC loss (p\0.05, R2 = 0.99). Our results indicate that the degradation of permafrost in CPCRW will result in a decrease in DOC and DON concentrations, a decline in DOC:DON, and a reduction in SUVA, possibly accompanied by

The role of dissolved organic matter bioavailability in promoting phytoplankton blooms in Florida Bay

The clear, shallow, oligotrophic waters of Florida Bay are characterized by low phytoplankton biomass, yet periodic cyanobacteria and diatom blooms do occur. We hypothesized that allochthonous dissolved organic matter (DOM) was providing a subsidy to the system in the form of bound nutrients. Water from four bay sites was incubated under natural light and dark conditions with enrichments of either DOM ( > 1 kD, 2×DOM) or inorganic nutrients (N+P). Samples were analyzed for bacterial numbers, bacterial production, phytoplankton biomass, phytoplankton community structure, and production, nutrients, and alkaline phosphatase (AP) activity. The influence of 2×DOM enrichment on phytoplankton biomass developed slowly during the incubations and was relatively small compared to nutrient additions. Inorganic nutrient additions resulted in an ephemeral bloom characterized initially as cyanobacterial and brown algae but which changed to dinoflagellate and/or brown algae by day six. The DIN:TP ratio decreased 10-fold in the N+P treatments as the system progressed towards N limitation. This ratio did not change significantly for 2×DOM treatments. In addition, these experiments indicated that both autotrophic and heterotrophic microbial populations in Florida Bay may fluctuate in their limitation by organic and inorganic nutrient availability. Both N+P and 2×DOM enrichments revealed significant and positive response in bioavailability of dissolved organic carbon (BDOC). Potential BDOC ranged from 1.1 to 35.5%, with the most labile forms occurring in Whipray Basin. BDOC at all sites was stimulated by the 2×DOM addition. Except for Duck Key, BDOC at all sites was also stimulated by the addition of N+P. BDOC was lower in the dry season than in the wet season (5.56% vs. 16.86%). This may be explained by the distinct chemical characteristics of the DOM produced at different times of year. Thus, both the heterotrophic and autotrophic microbial communities in Florida Bay are modulated by bioavailability of DOM. This has ramifications for the fate of DOM from the Everglades inputs, implicating DOM bioavailability as a contributing factor in regulating the onset, persistence, and composition of phytoplankton blooms.

Calculations on the end-group conformational barrier in carotenoids and a study of the bioavailability of xanthophyll esters in humans

The present study measures the increase in serum carotenoid concentration in 30 healthy individuals after supplementation with a low dose xanthophyll ester (3 and 6 mg of lutein equivalent/per day) when compared to a placebo. Serum levels of carotenoids were measured using HPLC and showed an increase in the concentration of lutein, zeaxanthin and four lutein metabolites proportional to dose. In order to further assess the importance of the end-group structure in carotenoids we have investigated the influence of the end-group type and functionality on the conformational energy barrier. We used the density functional method implemented on GAUSSIAN 98 to calculate the conformational energy curves for rotation of the P-ring or the E-ring relative to short polyene chains around the C6-C7 single bond. A large barrier is observed for the interconversion of conformers in the E-rings (8 kcal/mol) when compared to beta rings (2.3-3 kcal/mol).

Surfactant solubilization and subsequent bioavailability of phenanthrene

Contamination of soil, sediment and groundwater by hydrophobic organic compounds (HOCs) is a matter of growing concern because groundwater is a valuable and limited resource, and because such contamination is difficult to address. This investigation involved an experimental evaluation of the addition of several surfactant solutions to aqueous and soil-water systems contaminated with phenanthrene, a selected HOC. The results are presented in terms of: * phenanthrene solubilization achieved through surfactant addition * observed effects of surfactant addition on the mineralization of phenanthrene * estimation of relative toxicities of various surfactants using toxicity assays * literature-reported biodegradability/persistence of selected surfactants * surfactant sorption/precipitation onto soil and its impacts on proposed use of surfactant-amended remediation Surfactants were observed to facilitate the transfer of phenanthrene from the soil-sorbed phase to the aqueous pseudophase, however, surfactant solubilization did not translate into enhanced phenanthrene biodegradation.

Avaliação da pressão inspiratória nasal sniff na população obesa

Introdução: a avaliação da pressão inspiratória nasal (SNIP) é considerada uma manobra complementar da Pressão Inspiratória Máxima estática (PImax) em várias condições clínicas, porém não há relatos na obesidade. Por outro lado, a obesidade tem um importante impacto nos músculos respiratórios especialmente com maiores gordura abdominal o que provavelmente pode ser detectado na avaliação da SNIP que mensura mais precisamente a pressão diafragmática. Objetivo: analisar em obesos a relação entre SNIP e variáveis respiratórias e marcadores de adiposidade. Material e Método: num estudo transversal um total de 92 obesos (38.3±10.2 anos) sem história de doença respiratória ou cardíaca diagnosticada. Foram avaliados na espirometria (capacidade vital forçada-CVF; volume expiratório forçado no primeiro segundo-VEF1; volume de reserva expiratório-VRE) e pressões respiratórias estática (PImax, PEmax e SNIP) e dinâmica (ventilação voluntária máxima-VVM). Sendo considerados os marcadores de adiposidade: índice de adiposidade corporal-IAC; índice de massa corporal-IMC e circunferências do quadril (CQ), cintura (CC) e pescoço (CP). Resultados: 65 obesos mórbidos (IMC=50.8±8.1Kg/m2) e 27 obesos não mórbidos (IMC=35.6±2.7Kg/m2) foram homogêneos (p>0.05) na SNIP (99.1±24.5cmH2O, 87% do predito) e PImax (107.3±26.4cmH2O, 109% do predito). Existe correlação (r=0.5) entre SNIP e PImax somente no grupo de obesos mórbidos. De acordo com as correlações houve associação entre variáveis respiratórias (CVF r=0.48; VEF1 r=0.54; e VVM r=0.54), valores antropométricos (idade r=-0.44) e SNIP somente para os obesos mórbidos. Esses achados foram certificados quando também comparados a quantidade de gordura ao redor do pescoço (CP≥43cm). O modelo de regressão linear stepwise mostrou que a VVM parece ser o melhor preditor para explicar a SNIP nos obesos mórbidos. Nestes obesos a SNIP foi levemente mais baixa (87%predito) que os valores esperados para indivíduos brasileiros saudáveis. Conclusão: em obesos mórbidos a SNIP é moderadamente relacionada a PImax. A SNIP parece ser mais relacionada a VVM que à marcadores de adiposidade.

Oxygen tension, H2S, and NO bioavailability:is there an interaction?

Molecular oxygen (O2) is an essential component for survival and development. Variation in O2 levels leads to changes in molecular signaling and ultimately affects the physiological functions of many organisms. Nitric oxide (NO) and hydrogen sulfide (H2S) are two gaseous cellular signaling molecules that play key roles in several physiological functions involved in maintaining vascular homeostasis including vasodilation, anti-inflammation, and vascular growth. Apart from the aforementioned functions, NO and H2S are believed to mediate hypoxic responses and serve as O2 chemosensors in biological systems. In this literature review, we briefly discuss NO and H2S and their roles during hypoxia.

Effects of Natural Organic Matter on the Dissolution Kinetics and Bioavailability of Metal Oxide Nanoparticles

The rapid development of nanotechnology and wider applications of engineered nanomaterials (ENMs) in the last few decades have generated concerns regarding their environmental and health risks. After release into the environment, ENMs undergo aggregation, transformation, and, for metal-based nanomaterials, dissolution processes, which together determine their fate, bioavailability and toxicity to living organisms in the ecosystems. The rates of these processes are dependent on nanomaterial characteristics as well as complex environmental factors, including natural organic matter (NOM). As a ubiquitous component of aquatic systems, NOM plays a key role in the aggregation, dissolution and transformation of metal-based nanomaterials and colloids in aquatic environments.

The goal of this dissertation work is to investigate how NOM fractions with different chemical and molecular properties affect the dissolution kinetics of metal oxide ENMs, such as zinc oxide (ZnO) and copper oxide (CuO) nanoparticles (NPs), and consequently their bioavailability to aquatic vertebrate, with Gulf killifish (Fundulus grandis) embryos as model organisms.

ZnO NPs are known to dissolve at relatively fast rates, and the rate of dissolution is influenced by water chemistry, including the presence of Zn-chelating ligands. A challenge, however, remains in quantifying the dissolution of ZnO NPs, particularly for time scales that are short enough to determine rates. This dissertation assessed the application of anodic stripping voltammetry (ASV) with a hanging mercury drop electrode to directly measure the concentration of dissolved Zn in ZnO NP suspensions, without separation of the ZnO NPs from the aqueous phase. Dissolved zinc concentration measured by ASV ([Zn]ASV) was compared with that measured by inductively coupled plasma mass spectrometry (ICP-MS) after ultracentrifugation ([Zn]ICP-MS), for four types of ZnO NPs with different coatings and primary particle diameters. For small ZnO NPs (4-5 nm), [Zn]ASV was 20% higher than [Zn]ICP-MS, suggesting that these small NPs contributed to the voltammetric measurement. For larger ZnO NPs (approximately 20 nm), [Zn]ASV was (79±19)% of [Zn]ICP-MS, despite the high concentrations of ZnO NPs in suspension, suggesting that ASV can be used to accurately measure the dissolution kinetics of ZnO NPs of this primary particle size.

Using the ASV technique to directly measure dissolved zinc concentration, we examined the effects of 16 different NOM isolates on the dissolution kinetics of ZnO NPs in buffered potassium chloride solution. The observed dissolution rate constants (kobs) and dissolved zinc concentrations at equilibrium increased linearly with NOM concentration (from 0 to 40 mg-C L-1) for Suwannee River humic acid (SRHA), Suwannee River fulvic acid and Pony Lake fulvic acid. When dissolution rates were compared for the 16 NOM isolates, kobs was positively correlated with certain properties of NOM, including specific ultraviolet absorbance (SUVA), aromatic and carbonyl carbon contents, and molecular weight. Dissolution rate constants were negatively correlated to hydrogen/carbon ratio and aliphatic carbon content. The observed correlations indicate that aromatic carbon content is a key factor in determining the rate of NOM-promoted dissolution of ZnO NPs. NOM isolates with higher SUVA were also more effective at enhancing the colloidal stability of the NPs; however, the NOM-promoted dissolution was likely due to enhanced interactions between surface metal ions and NOM rather than smaller aggregate size.

Based on the above results, we designed experiments to quantitatively link the dissolution kinetics and bioavailability of CuO NPs to Gulf killifish embryos under the influence of NOM. The CuO NPs dissolved to varying degrees and at different rates in diluted 5‰ artificial seawater buffered to different pH (6.3-7.5), with or without selected NOM isolates at various concentrations (0.1-10 mg-C L-1). NOM isolates with higher SUVA and aromatic carbon content (such as SRHA) were more effective at promoting the dissolution of CuO NPs, as with ZnO NPs, especially at higher NOM concentrations. On the other hand, the presence of NOM decreased the bioavailability of dissolved Cu ions, with the uptake rate constant negatively correlated to dissolved organic carbon concentration ([DOC]) multiplied by SUVA, a combined parameter indicative of aromatic carbon concentration in the media. When the embryos were exposed to CuO NP suspension, changes in their Cu content were due to the uptake of both dissolved Cu ions and nanoparticulate CuO. The uptake rate constant of nanoparticulate CuO was also negatively correlated to [DOC]×SUVA, in a fashion roughly proportional to changes in dissolved Cu uptake rate constant. Thus, the ratio of uptake rate constants from dissolved Cu and nanoparticulate CuO (ranging from 12 to 22, on average 17±4) were insensitive to NOM type or concentration. Instead, the relative contributions of these two Cu forms were largely determined by the percentage of CuO NP that was dissolved.

Overall, this dissertation elucidated the important role that dissolved NOM plays in affecting the environmental fate and bioavailability of soluble metal-based nanomaterials. This dissertation work identified aromatic carbon content and its indicator SUVA as key NOM properties that influence the dissolution, aggregation and biouptake kinetics of metal oxide NPs and highlighted dissolution rate as a useful functional assay for assessing the relative contributions of dissolved and nanoparticulate forms to metal bioavailability. Findings of this dissertation work will be helpful for predicting the environmental risks of engineered nanomaterials.

Discerning the sub-phonemic identity of pre-nasal /æ/ in British Columbia English

There are two features of /æ/ in British Columbia (BC) English that are widely attested in the literature: it is undergoing retraction and lowering and it is sensitive to the influence of certain following consonants. The present study aims to utilize both features to evaluate the phonological status of /æ/ before nasal consonants in BC English by examining the progression of sound change and the phonemic organization of /æ/ in different environments. Specifically, production and perception results are taken together to evaluate the phonetic position of pre-nasal /æ/ relative to other environments. These results are interpreted within a modular feedforward architecture of phonology to establish the phonological (allophonic) and phonetic (shallowphonic) rules that govern the internal relationships between the subphonemic elements of /æ/ in BC English. Further, the findings of this study provide evidence for the allophone being the target of sound change, rather than the phoneme.