The present work describes the development of a method for the estimation of plasma kinin and kininogen suitable for use with clinical blood samples. This method has been used to study kinin-release in two clinical conditions, the carcinoid and post-gastrectomy (dumping) syndromes, and its possible relationship to physiological stress. The experimental study is divided into two sections, (i) Methodology and (ii) Application of Method.

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The aims of this study tested the hypothesis that expression of heparin-binding growth factors (HBGFs) in normal placental development was altered in a specific pregnancy disorder preeclampsia. HBGFs bind to heparin, a glycosaminoglycan (GAG) affecting activity. I investigated the role of heparin and HBGFs in pathophysiology of pre-eclampsia. Placental tissue from a cohort study of 87 women was performed following uncomplicated pregnancy at term, but not in labour (TNL, n=26), preterm labour (PTL, n=17), following labour onset (TL, n=21), first trimester (FNL, n=4) and pre-eclampsia (PE, n=19). The HBGFs studied were vascular endothelial growth factor (VEGF), placental growth factor (PLGF), fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), heparin-binding epidermal growth factor (HB-EGF), midkine (MK), pleiotrophin (PTN), and cluster differentiation (CD105). The localisation of HBGFs and receptors VEGFR-1, /(sflt-1), PLGFR-1, VEGFR-2 and FGF2R-1 in placenta were detected. The expression of VEGF, PLGF, FGF2, HGF, PDGF, CD105 was confined to villous trophoblast, endothelial cells except for MK, HB-EGF and PTN was specifically to villous trophoblast. The total RNA production in human placentae samples (n=7) from PE and controls were analysed using qRTPCR. Placental expression of mRNA was extracted for primer assays of PLGF, FGF2, MK, PTN, and endogenous housekeeping gene as Succinate dehydrogenase complex subunit A (SDHA). FGF2 and SDHA mRNA expression was significantly different using Mann-Whitney U test. An in vitro villous trophoblast invasion model was performed with human fibrosarcoma HT1080 invasive cells (positive control), mouse embryonic fibroblast NIH/3T3 non-invasive cells (negative control) and immortalised human primary villous trophoblastic cell lines TCL-1.The greatest stimulation was by FGF2, PDGF-BB, HGF, MK and co-incubation with heparin enhanced these responses, except for PTN using the Mann-Whitney U test. Heparin’s role is indicated in mediating the effects of HBGFs. It’s suggests heparin therapeutic use in the treatment of pre-eclampsia.

In recent decades, research has unravelled fascinating detail about the molecular mechanisms underpinning pharmacologic loss of consciousness (LOC). However, the systems-level mechanisms are far less clear. Recent genetic approaches, however, enable unprecedented dissection on neural pathways, and they are paving a way for this line of research. The focus of this thesis is to investigate the neuroanatomical substrates of commonly used drugs which reversibly render us unconscious. Zolpidem is a positive allosteric modulator (PAM) of the GABAA receptor which binds to the benzodiazepine (BZ) site. Because zolpidem binds α1-3,β,γ2 containing GABAA receptors, which are widespread, it acts virtually everywhere. We do not know if zolpidem causes sleep by enhancing GABAergic inhibition throughout the entire brain, or if the therapeutic sleep-inducing property depends upon specific brain circuitry. γ2I77 mice are devoid of zolpidem-sensitivity. But, zolpidem-sensitivity can be restored selectively in brain regions, enabling dissection of the circuitry involved in zolpidem's effect. To isolate the therapeutic effect of zolpidem we deleted GABAA-γ2I77-subunits and replaced them with GABAA-γ2F77-subunits in HDC neurons or frontal-cortex in isolation. We were able to selectively restore zolpidem-sensitivity in target neurons. This conferred zolpidem-enhanced IPSCs locally. Compared with wild-type mice and zolpidem-insensitive γ2I77lox mice, we found that GABAA-γ2F77 receptors in either HDC-neurons or frontal cortex alone were enough to rescue the majority of zolpidem-mediated sleep. The response in HDC-γ2F77 mice was similar to that of an H1-receptor antagonist. By producing a null effect in a negative-control area - the superior colliculus - we show that HDC neurons and the frontal cortex are both substrates involved in zolpidem-mediated sleep. We also investigated the role of synaptic-inhibition onto corticothalamic-neurons in anaesthetic-induced LOC and sleep-wake. To do this, we genetically ablated γ2-subunits from layer-6 corticothalamic-cells by crossing Ntsr1-Cre mice with GABAA-γ2I77lox mice. We found this reduced isoflurane sensitivity, but left sleep-wake behaviours virtually unaffected.

Inflammation is thought to play a driving role in an expanding number of vascular and cerebral pathologies. Sulforaphane (SFN) is a naturally occurring isothiocyanate which has previously been shown to harbour anti-inflammatory properties in several immune cell types. In this work, intra vital microscopy was used to visualise the cellular interactions within the cerebral microvasculature stimulated downstream of an inflammatory challenge, namely intraperitoneal injection of LPS (0.1-4mg/kg). This resulted in a time, not dose, dependent stimulation of leukocyte-endothelial interactions; the number of rolling cells significantly increased, cells exhibited a slow rolling velocity and the number of adherent cells significantly increased versus sham or vehicle treated animals. SFN pre-treatment at 50mg/kg 24hr prior to LPS challenge (0.5mg/kg), increased the number of cells rolling through the cerebral microvasculature, significantly increased the speed at which they were rolling versus LPS treated animals and decreased the number of cells that were adherent to the pial vessel walls. These alterations were not associated with alterations in systemic cytokine levels (IL12p70, IL-6, TNFα, IL-10, IFNγ, MCP-1) or nuclear Nrf2 expression within the cerebral cortex. LPS (1µg/ml) increased IL-1β release from isolated human neutrophils which also showed no alteration following SFN incubation (0.1-25µM). Investigation into the effects of SFN revealed that SFN significantly altered the ability of platelets to aggregation in response to several agonists (thrombin, collagen and ADP), and that this response was not associated with the intracellular Ca2+ movement or induction of LDH release. This work highlights the ability of SFN to alter inflammatory cell interactions within the cerebral microvasculature in vivo, and also indicates the inhibitory effect of SFN on isolated platelet function ex vivo.

The problems of non-steroidal anti-inflammatory drugs (NSAIDs) as environmental contaminants is an area of concern. NSAIDs are heavily relied upon to treat pain and inflammation. With such prevalence, these compounds are now entering the environment via many routes, such as water discharge and contaminated food. This results in subsequent exposure and effects on various animal species. One such example is diclofenac, which was associated with the extinction of Gyps vultures in Asia. The detection of diclofenac was based on post-mortem samples i.e. after a large decline in populations. In this research non-invasive samples i.e hairs and feathers are analysed pre-mortality as a preventive measure for early detection. A simultaneous liquid chromatography-mass spectrometry (LC-MS) method for detection of eighteen compounds, either of known toxicological effects or future threat (NSAIDs - aceclofenac, carprofen, diclofenac, flunixin, ketoprofen, mefenamic acid, meloxicam, nimesulide, phenylbutazone, piroxicam and suxibuzone; metabolites - oxyphenylbutazone, 3-hydroxymethyl mefenamic acid, 4-hydroxydiclofenac, 4-hydroxynimesulide, 5-carboxymeloxicam, 5-hydroxyflunixin and 5-hydroxypiroxicam) has been developed and validated. A newly optimised sample preparation method was applied to hairs/feathers. Precision of the analytical method was within 10% relative standard deviations for the majority of compounds. Recoveries averaged 83% and limits of detection (LOD) ranged 0.01 to 0.2μg/g. For diclofenac, flunixin, mefenamic acid, oxyphenylbutazone, piroxicam and 5-hydroxyflunixin, LODs were lower than previously reported. Various animal hairs/feathers were analysed (n=20) and in two samples piroxicam and phenylbutazone were individually detected, at 1.2μg/g ± 0.002 and 1.8μg/g ± 0.011 respectively. The LC-MS method reported here has been validated for the first time using animal hair/feather samples. This range of NSAIDs and metabolites have never been reported before. LODs and LOQs of metabolites are reported for the first time. The detection of piroxicam and phenylbutazone in feathers highlights the viability of testing keratinous matrices.

Increased use of complex cellular systems to study hepatic drug disposition in vitro has been associated with increased complexity of models applied for data analysis. Despite these advances, a tendency to under-predict transporter-mediated hepatic clearance using in vitro data is generally reported. The aim of the current project was to investigate systematically the reasons for this under-prediction. Focus of the work was on differences in quantitative expression between Organic anion-transporting polypeptide (OATP) transporters in the in vitro cellular systems and human liver tissue and their impact on physiologically-based pharmacokinetic-in vitro-in vivo extrapolation (IVIVE) of transporter kinetic data. In addition, various modelling techniques to estimate kinetic parameters were explored, using existing and newly generated data in rat and human plated hepatocytes. Finally, to develop a novel experimental method for investigating uptake in a reduced experimental format, validate the method in rat hepatocytes and apply it to human and investigate the impact on IVIVE of transporter kinetic data. Nine OATP substrates were selected, namely rosuvastatin, valsartan, repaglinide, olmesartan acid, candesartan acid, fexofenadine, cerivastatin, glibenclamide and rosiglitazone. Meta-analysis of reported quantitative protein levels of OATP transporters in different human hepatocyte formats (suspended and sandwich-cultured hepatocytes) and liver tissue was performed. The analysis showed maximal 2.5-fold difference between OATP expression in hepatocytes and liver tissue (data from 86 individuals) which could not solely rationalize under-prediction of active uptake clearance reported so far. Weak correlation between OATP1B1 expression and age was noted, but no direct relationship between sex and OATP expression levels was observed. A new modelling strategy for fitting sparse rosuvastatin in vitro uptake data from human hepatocytes was explored. A mechanistic two-compartment model was set up NONMEM 7.3 allowing the simultaneous estimation of rosuvastatin kinetic parameters describing transported-mediated uptake (CLactive), bidirectional passive diffusion (CLpassive) and intracellular binding (fu,cell) from uptake experiments carried out over 2 to 60 min and over a range of concentrations (0.1 to 300 µM). Use of a population-based approach resulted in structural and stochastic parameters estimated with confidence using data generated from different experimental designs in human hepatocytes. Kinetic parameters were determined as species-dependent with the exception of Michaelis-Menten constant. In addition, a novel experimental and mechanistic modelling approach was developed using freshly isolated rat hepatocytes; method was subsequently applied to assess active uptake in plated cryopreserved human hepatocytes. Modelling and simulation was applied to optimise the experimental design conducted at a single concentration of 1 µM and over extended incubation time. The novel experimental protocol comprised of an uptake phase until initial equilibrium between cell and medium was reached, followed by a post-wash phase. The optimal post-wash medium, the stability of uptake transporters over time and the cell loss throughout the experiment were investigated. When required, the mechanistic compartmental model was extended to assess metabolism (repaglinide, cerivastatin, glibenclamide and rosiglitazone). The analysis of generated data allowed a good definition of the cellular concentration-time profiles of selected drugs and estimation of the kinetic parameters with precision. The decline in post-wash cellular concentrations was related to permeability properties, extent of intracellular binding and ratio of active clearance over passive diffusion of drugs investigated. Active process was a major contributor to the total uptake, ranging from 58-99.5 % for cerivastatin and fexofenadine in rat and from 62 % to 99.6 % for repaglinide and valsartan in human hepatocytes (FOP). In addition, the application of the novel design allowed clear differentiation of the cellular processes and reduced previously observed correlation between CLpassive and fu,cell. In rat, CLactive was on average 4.8-fold greater compared to human hepatocytes. CLactive was independent of SLCO1B1 c.521 genotype for rosuvastatin, valsartan and repaglinide (data from 4 donors). Direct comparison to data previously reported in literature in the same donor of human hepatocytes (HU4199) showed significant improvement in prediction of hepatic intrinsic clearance and reduction in under-prediction trend and need for empirical scaling factors for model drugs repaglinide, valsartan and rosuvastatin.

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