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Dielectrophoresis (DEP), the induced motion of cells in an inhomogeneous electric field, can be measured by studying the motion of the cells at different frequencies. This work uses a light absorption method to determine the DEP force and extract the dielectric properties of cell populations. Previously, light intensity changes in a 3D well electrode have been measured with a microscope and a single channel signal generator. This work sets out to enable real ~time measurement of cell populations by implementing a digital camera, lens and light source alongside a 20 channel signal generator in one machine, the DEP well instrument, to measure light intensity changes for 20 wells simultaneously. Optimisation of the optics is carried out through the evaluation of different cameras, lenses and light sources and measuring the light intensity changes of yeast populations in 4 wells that are energized with the same frequency, in order to ensure that the system is able to give consistent results. Finally, the application of the DEP well instrument for detection of drug cytotoxicity and rapid detection of apoptosis, both important in the development of new chemotherapeutic drugs, was studied. Both apoptosis and cytotoxicity of drugs can be characterized by changes in the membrane and cytoplasm properties of cells resulting in various distinct sub-populations within a sample. Rapid detection of apoptosis was examined by inducing HeLa cells with the well known chemotherapeutic agent staurosporine and comparing the results obtained with DEP and those obtained with the gold standard method of measuring apoptosis, Annexin V assay with flow cytometry. The ability of the DEP well instrument to test drug cytotoxicity on suspension and adherent cells was studied by inducing cells with the chemotherapeutic agent doxorubicin and measuring cell viability with DEP after different incubation times. The results were compared with the calorimetric assay 3-(4,S-Dimethylthiazol-2-yl)-2,S-Diphenyltetrazolium Bromide (MTT) and trypan blue experimental results, as well as with previously published values

Since the early 1990's, average bioequivalence studies have served as the international standard for demonstrating that two formulations of drug product will provide the same therapeutic benefit and safety profile when used in the marketplace. Population (PBE) and Individual (IBE) bioequivalence have been the subject of intense international debate since methods for their assessment were proposed in the late 1980's. Guidance has been proposed by the Food and Drug Administration of the United States government for the implementation of these techniques in the pioneer and generic pharmaceutical industries. As of the present time, no consensus among regulators, academia, and industry has been established. The need for more stringent population and individual bioequivalence has not been demonstrated, and it is known that the criteria proposed by FDA are actually less stringent under certain conditions. The properties of method-of-moments and restricted maximum likelihood modelling in replicate designs will be explored in Chapter 2, and the application of these techniques in the assessment of average bioequivalence will be considered. Individual and population bioequivalence criteria in replicate cross-over designs will be explored in Chapters 3 and 4, respectively, and retrospective data analysis will be used to characterise the properties and behaviour of the metrics. Simulation experiments will be conducted in Chapter 5 to address questions arising from the retrospective data analyses in Chapters 2 through 4. Additionally, simulation will be used to explore of a potential phenomenon known as 'bio-creep' - that is the transitivity of individual bioequivalence in practice. Another bioequivalence problem is then considered to conclude the thesis; that of compaxing rate and extent of exposure between differing ethnic groups as described in ICH-E5 (1998). The properties of the population bioequivalence metric and an alternative metric will be characterised in small and large samples from parallel group studies. Inference will be illustrated using data from a recent submission and simulation studies.

In vitro epithelial cell cultures are increasingly used to model drug permeability, as predictive tools for absorption in humans. Medical regulatory agencies recommend in vitro permeability screening for biopharmaceutical classification of novel therapeutic compounds, and recently published guidelines on investigating interactions of novel therapeutic compounds with clinically relevant transporters. The expression and functionality of drug transporters in the lung is poorly characterised, and insufficient to allow detailed understanding of drug-transporter interactions in the airways. Additionally, as human in vitro permeability is used to predict absorption from rat in vivo, a rat bronchial epithelium in vitro cell line would aid the understanding of interspecies differences in transporter-mediated drug trafficking. This thesis investigates the morphological and physiological barrier properties of Calu-3, normal human bronchial epithelial (NHBE) cell layers and rat airway epithelial cell (RL-65) cultures. The morphology and barrier integrity of RL-65 layers were shown to be in agreement with existing human bronchial epithelial cell models after culture for 8 days at air-liquid interface. The expression of >30 ABC, SLC and SLCO transporters in human models was in general agreement with published expression levels in human lungs. MDR1 functionality was investigated, and whilst no asymmetric transport of 3H-digoxin was observed in RL-65 cell layers, net secretory transport was observed for Calu-3 cell layers at both low (25-30) and high (45-45) passage number and for some batches of NHBE cell layers. Chemical, metabolic and biological inhibitors were employed to evaluate MDR1 contribution to 3H-digoxin trafficking, however the exact transporter(s) involved could not be determined. Whilst MDR1 functionality could not be ruled out, results suggest that it is unlikely to be the main transporter involved in 3H-digoxin trafficking in the bronchial epithelium. These studies have highlighted the need for more specific approaches to investigating transporter functionality in in vitro systems.