Glucocorticoids (GCs) are widely used to treat inflammatory diseases of the gut and liver. However, they are ineffective in up to a third of patients. In ulcerative colitis (UC) such GC resistant individuals require a colectomy, while patients with severe alcoholic hepatitis (AH) develop fatal progressive liver failure. Early stratification of these high-risk patients is critical to improve our understanding of the mechanisms of GC resistance and also for the development of better therapies. The work presented in this thesis aimed to extend the application of a candidate biomarker of GC resistant disease from UC to AH. Based on this, differential lymphocyte responses to GCs were then interrogated, and the effect of interleukin-2 on GC responsiveness assessed. An evaluation of the role of different T helper (Th) cell subsets in driving the GC resistant phenotype was then conducted, followed by an investigation of the role of monocytes in shaping these T cell responses to GCs. These studies have confirmed that the in vitro effect of GCs on lymphocyte proliferation corresponds with clinical outcome in AH as well as UC, highlighting the role of T cell responses. Blockade of IL-2 improved GC sensitivity in vitro and investigation of intracellular signalling pathways demonstrated that there was impaired GC regulation of the pro-inflammatory transcription factor NFKB in GC resistant cells. NFKB can induce Th17 cell differentiation and IL-17 expression was found to be refractory to GC suppression in CD4+ T cells. Toll-like receptors (TLRs) were then shown to influence monocyte-mediated CD4+ T cell differentiation in both man (in vitro) and mouse (in vivo). Monocyte subsets were critical in determining this T cell fate, and innate immune responses are therefore a key potential driver of the GC resistant phenotype. This work has led to the development of a protocol for a Phase II clinical trial of IL-2 blockade in GC resistant AH and also further mechanistic studies to evaluate the role of monocytes in GC resistant diseases.

The plant Levisticum officinale (Lovage) is commonly used as a condiment. It is traditionally used as an anti-flatulent, anti-dyspepsic, spasmolytic and anti-inflammatory agent to relieve rheumatic pain. The main reason for examining Lovage in this study was to determine its potential anti-inflammatory 1 properties in human monocytes (THP-1 cells) stimulated with lipopolysaccharide (LPS). Solvent extracts (methanol/water 1:1 and hexane) of Lovage leaves were used in this study.

Proteinase activated receptor-2 (PAR-2) is a novel G-protein coupled receptor, that is activated by means of proteolytic cleavage (Macfarlane et al, 2001) and has both pro and anti-inflammatory actions depending upon the system examined. PAR-2 have been linked to the stress-activated protein kinases (SAPKs), JNK and p38 MAP kinase and NFK13 signalling (Kanke et al, 2001; Sabri et al, 2000), pathways known to be involved in proinflammatory responses in several cell types. TNFa has been demonstrated to up-regulate PAR-2 expression in a variety of cell types (Nystedt et al, 1996; Ritchie et al, 2007). Since both TNFa and PAR-2 are implicated in inflammation, we examined the possibility of altered PAR-2 trafficking under inflammatory conditions and possible crosstalk between PAR-2 and TNFa at the level of intracellular signalling. Previous studies have characterised P AR-2 trafficking in transfected cell lines, however the effects of inflammatory stimuli on the kinetics of PAR-2 trafficking has not been investigated. The study sought to re-characterise PAR-2 trafficking in the presence of inflammatory stimuli. NCTC2544 cells transfected with YFP epitope tagged PAR-2, demonstrated clear PAR-2 expression and trafficking of the receptor was successfully characterised, however no significant differences in the kinetics of P AR-2 trafficking under inflammatory conditions compared to control was observed. The latter part of the study examined PAR-2 and TNFa mediated activation of the MAPK and NFK13 pathways. In a keratinocyte cell line stably expressing PAR-2 (CloneG), trypsin, SLIGKV-OH, and TNFa, caused a time and concentration-dependent increase in p38 MAPK and JNK phosphorylation however, preliminary results failed to show evidence of synergy between the receptors. Surprisingly however, pre-activation of P AR-2 substantially reduced the ability of TNFa to activate JNK. The inhibitory effect of P AR-2 was mimicked by the protein kinase C activator PMA, partially reversed by the PKC inhibitor GF109203X, and completely reversed by the novel Gaqlll inhibitor YM-254890, consistent with a role for both Ca2+ -dependent and independent PKC isoforms and for P AR-2 coupling to Gaq/11 to mediate this agonist driven inhibitory response. These results indicate a potential mechanistic explanation for both the anti and pro-inflammatory actions of P AR- 2, and highlight a possible novel therapeutic avenue for the development ofPAR-2 agonists as anti-inflammatory drugs.

For six decades tetracyclines have been successfully used for their broad spectrum antibiotic effects. However, non-antibiotic effects of tetracyclines have been reported. The anti-inflammatory effects of tetracycline drugs have been investigated in the context of a range of inflammatory diseases including sepsis and a number of neurodegenerative diseases. This thesis investigates the effects of a range of clinically important tetracyclines (oxytetracycline, doxycycline, minocycline and tigecycline) on the ability of the J774.2 cell line to produce nitric oxide when stimulated with the bacterial cell wall component, LPS.

Erythropoietin (EPO) is an erythropoietic cytokine that is also neuroprotective in vitro and in vivo. Neither the mechanism of action of EPO in neuroprotectio, nor the receptor involved is completely known. In fact, EPO regulates erythropoiesis by the homodimeric EPO receptor (EPOR)2. Variants of EPO, not binding (EPOR)2, are still neuroprotective, therefore another receptor may mediate this effect. In vivo, EPO is anti-inflammatory in several models of disease but, to date, a direct anti- inflammatory effect in vitro has not been clearly found. The focus of this thesis work was a twofold. Firstly, a direct anti-inflammatory effect of EPO was investigated in vitro. It was confirmed that EPO did not have any effect on production of cytokines induced by LPS. In addition, EPO did not reduce cytokines induced by alarmins and other inflammatory stimuli. EPO did not inhibit the pro-inflammatory receptor TREM-1. Finally, EPO did not act as anti-inflammatory by mobilization of endothelial precursor cells in vivo. The second focus of this thesis work was the study of a possible role of EPO on myelination by analysing the induction of myelin genes during differentiation of an oligodendrocyte cell line. EPO upregulated myelin gene expression (MaG and MBP), as studied by qPCR and Western Blot. EPOR was required for the effect of EPO, observed only in cells overexpressing EPOR. EPO induced high levels of the early growth response gene EGR2 that was however not involved in myelin gene induction. Finally, EPO was unable to induce myelin genes in an in vivo model of demyelination induced by cuprizone, neither at the peak of demyelination (3 and 5 weeks) nor during the recovery phase. Greater understanding of effects and mechanisms of action of EPO in the CNS would be useful to find new therapies promoting repair, for instance in diseases like MS in which no drug is available for that purpose.

Co-crystals are multi-component, single phase materials in which the co-crystal formers exist in an uncharged state. This interaction between components occurs without affecting the intra-molecular covalent bonding of the involved components or altering their chemical integrity. This class of materials has recently gained the interest in the pharmaceutical industry for modifying the physico-chemical properties of some active pharmaceutical ingredients (APIs) such as solubility, hygroscopicity, and mechanical properties. However like all multi-component systems, there are some analytical challenges associated with using traditional quality control (QC) tools that involve sample preparation steps prior to the analysis. This can sometimes have an impact on the physical state of such systems and therefore affect the outcomes of analysis correlated with the actual materials. Flurbiprofen (FBP) and nicotinamide (NCT) in this work were selected to form a model co-crystal system. FBP falls in the carboxylated group of non steroidal anti-inflammatory drugs (NSAIDs), used herein as an API. It belongs to class II of the Biopharmaceutics Classification System (BCS), therefore its low aqueous solubility and dissolution rate affect its bioavailability. NCT is a vitamin B3 derivative and generally regarded as safe (GRAS) substance. It is used as co-crystallising agent due to its solubility enhancing property. FBP-NCT co-crystal system was previously studied by Berry et al., where a FBP-NCT co-crystal was prepared using Kofler method and screened by means of the hot-stage microscopy (HSM). FBP-NCT co-crystal was recently prepared via rapid evaporation from ethanol solution, and screened for its physicochemical and mechanical properties by Shing et al. who found an improvement in such properties compared to the pure API (FBP). There is a wide range of methods for preparing co-crystals, of which methodologies with minimal environment impact were mainly adopted in this work i.e., Ko er fusion and co-grinding methods. Within the Kofler fusion method, it was feasible to identify and screen simultaneously parent components as well as the emerging material at room temperature (RT) by means of confocal Raman microscopy. Detailed information from Raman mapping on the investigated phases were achieved using statistical analysis. The analysis resulted in a refinement regarding the traditional assumption about the binary phase diagram. It also revealed the presence of two forms of FBP-NCT co-crystals which were consequently explored by coupling thermal analysis with Raman spectroscopy and X-ray diffraction. Using co-grinding preparation, FBP-NCT co-crystals were prepared on a large scale; enough for solid dosage formulation. Subsequent crystallisation of the resulting compound using a solution method generated single crystals suitable for x-ray crystal structure determination. Moreover in this work, a solid-state dosage form of FBP-NCT co-crystal was prepared for the first time, then investigated using transmission Raman spectroscopy. The investigation included the presence of drug and excipient, and their composition in tablets. This procedure can be considered as a platform for studying the QC of drug preparation, using a reliable, non-destructive, non-invasive, and very rapid analytical tool. Such type of study complied well with the food and drug administrative (FDA) outlines on employing process analytical technology (PAT) protocol for analysing and controlling pharmaceutical manufacturing processes.