This thesis tests the hypothesis that certain mediators (purines, catecholamines and bradykinin), which are known to be involved in inflammation, contribute to the sensitisation of peripheral nociceptors that occurs in chronic inflammation and hyperalgesia associated with arthritis. The major part of the studies determined the role of these substances and their pharmacological receptors in modulating discharge recorded from high threshold C-fibre mechanonociceptors (on-going or 'spontaneous', and mechanically-evoked) in normal ankle joints and in those with a monoarthritis induced by Freunds complete adjuvant (FCA). Some of the <I>in-vitro</I> neuropharmacological investigations were complemented by behavioural studies on intact normal and arthritic rats. Related <I>in vitro</I> experiments were performed involving a) extracellular 'grease gap' recordings from various peripheral nerves and b) contractile responses of the electrically-stimulated rat vas deferens. Recordings from C-fibres in arthritic joints revealed an enhanced resting discharge, a greater number of receptive fields and lower mechanical activation thresholds (sensitisation) of mechanonociceptors as compared to untreated joints. Indomethacin significantly attenuated, but did not abolish, either the elevated resting discharges recorded from C-fibres in arthritic joints or the swelling, mechanical hyperalgesia and inflammation associated with functional studies in monoarthric rats. The results show that C-fibre afferent discharge from articular mechanonociceptors in the normal and chronically-inflamed (adjuvant-arthritic) rat ankle joint is modulated by bradykinin (excitation and sensitisation). Catecholamines can also cause excitation and sensitisation of arthritic joints, but purines appear to have no effect on the activity of these sensory receptors. Bradykinin, acting via bradykinin B<SUB>2</SUB> receptors plays an important role in altering neural excitability in the rat, and different subtypes of B<SUB>2</SUB> receptor may be involved. Overall, the present results add further to our knowledge and understanding of the peripheral mechanisms involved in nociception in the normal state and in chronic inflammation.

The aims of this study were to examine the role of oestrogen in a series of ER-positive ovarian cancer cell line models, including the first characterised lines with moderate-high levels of ER. The effects of oestrogen on growth were initially determined in nine ovarian cell lines possessing a range of ER from 0 to 132 fmol/mg protein. Sensitivity to oestrogen correlated with the level of ER expression in that concentrations of 17 β-oestradiol (E<SUB>2</SUB>) between 10<SUP>-12</SUP> and 10<SUP>-6</SUP>M stimulated the growth of the PEO1, PEO4, and PEO6 cell lines which possess moderate-high levels of receptors between 96 and 132 fmol/mg protein, whereas there was no change in the growth of the PEA1 and PEA2 cell lines which have an ER content of less than 30 fmol/mg protein. The growth of the ER negative lines PEO14, PEO16 and PE023 was also unchanged by E<SUB>2</SUB> treatment. Concentrations of E<SUB>2</SUB> which were stimulatory to the PEO1 cell line were inhibitory to a cisplatin-resistant derivative, PEO1<SUP>CDDP</SUP> despite this also possessing moderate-high levels of ER. Effects of E<SUB>2</SUB> on growth were also examined in two ovarian xenograft models grown in nude mice; PEO4 and HOX60 which are ER-positive and ER-negative respectively. Exposure of the xenografts to a subcutaneously planted oestrogen pellet produced a significant inhibition in PEO4 growth but no difference in HOX60 growth as compared to controls. If these results reflect the clinical situation, then there is a valid case for the use of anti-oestrogen therapy in a subset of patients with ovarian tumours expressing moderate-high levels of ER. Agents which interfere with the TGF-α and IGF-mediated growth pathways may also have therapeutic benefit.

The aim of this study was to investigate the effects of an <I>in utero</I> hypoxic insult on the near-term foetal rat brain. An hypoxic insult of 10, 20 or 30 minutes was created by clamping the utero-placental vessels of one uterine horn in an anaesthetised pregnant Lister hooded rat at 22 days gestation. After completion of the occlusion period the dam was killed and the foetal rats were delivered by Caesarian section and resuscitated. Alternatively the clamps were removed, the uterine horn placed back inside the dam and the wound sutured. After a recovery period of 20 minutes, during which time the dam was not anaesthetised, the dam was killed and the foetal rats were delivered by Caesarian section and resuscitated. Biochemical analysis of homogenised and deproteinised brain tissue samples from the neonatal rats showed a level of metabolic deficit typically associated with irreversible neuronal necrosis in adult models of hypoxicischaemia. The level of brain tissue in rats subjected to a 30 minute clamp without recovery was 19.77± 0.8nmols/mg brain tissue whereas in control rats the level of lactate was 7.98 ± 0.2nmols/mg brain tissue (p < 0.001). Brain tissue ATP declined by more than 80% from 2.67 ± 0.2nmols/mg brain tissue in control rats to 0.59 ± 0.1nmols/mg brain tissue in hypoxic rats (p < 0.001). Other metabolites also showed changes consistent with a major hypoxic insult. This study has shown that despite a significant metabolic deficit those rats which survive an <I>in utero</I> hypoxic insult suffer only limited and discrete neuropathological changes and no apparent long-term physiological or cognitive deficits. These results suggest that the near-term foetal rat brain has a high level of resistance to the long term consequences of a severe hypoxic insult.

In this thesis, I have employed the γ-glutamyl prodrug approach to targeting drugs to the kidney to explore the renal formation and actions of 5-HT in healthy human subjects. I compared the effects of intravenous infusions of 5-hydroxy-L-tryptophan (L-5-HTP), the immediate precursor of 5-HT, and γ-L-glutamyl-5-hydroxy-L-tryptophan (glu-5-HTP), the glutamyl derivative of L-5-HTP. There were marked increases in the urinary excretion of 5-HT, without changes in circulating 5-HT levels measured in platelet-rich plasma, consistent with intrarenal synthesis of 5-HT. Both compounds reduced urinary sodium excretion in the absence of significant alterations in renal haemodynamics suggesting a predominantly tubular effect on sodium reabsorption. The urinary metabolite data and reduced extrarenal effects of glu-5-HTP, as evidenced by changes in growth hormone and aldosterone release, blood pressure and incidence of adverse effects, suggested that the glutamyl prodrug was relatively more selective for the kidney than L-5-HTP. Carbidopa produced a 99% reduction in urinary excretion of 5-HT after glu-5-HTP and abolished its antinatriuretic effect. These observations indicated that glu-5-HTP, or its intermediate metabolite L-5-HTP, had no effect <I>per se</I> and required conversion to 5-HT to exert an effect on sodium excretion. I compared the relative effectiveness of γ-L-glutamyl-L -tryptophan (glu-TRP), the glutamyl derivative of L-tryptophan, and glu-5-HTP as substrates for 5-HT synthesis in the kidney. Glu-TRP, unlike glu-5-HTP, failed to increase the urinary excretion of 5-HTP or 5-HT and to reduce sodium excretion suggesting an absence of effective renal synthesis of 5-HT following glu-TRP infusion. Glu-5-HTP could be a valuable experimental tool for manipulating renal 5-HT production and exploring the renal formation and effects of 5-HT in man in health and disease.

In recent years, pharmaceutical research has begun to shift from the development of small molecule chemical entities, to complex high value, low volume biologics. The cost of batch losses or product recall due to the presence of microbial, chemical or physical contaminants can have serious implications for a business. It is therefore important that as the complexity of pharmaceutical products increases, the techniques utilised in the analysis and characterisation of these products and their excipients also moves forward, thereby ensuring product quality and patient safety. This research explores three areas of analysis and characterisation relating to pharmaceutical products: microbial testing; oxidative stability testing and container integrity testing. The detection and identification of microbiological contamination using traditional microbial methods (TMMs) have a number of limitations such as long testing times and reliance on subjective assessment leading to operator error. More recently developed rapid microbial methods (RMMs) have been designed to overcome these limitations and their uptake would enable microbial testing to align with the process analytical technology (PAT) approach to process monitoring and control. A review of available techniques and those implemented by a large pharmaceutical company, GlaxoSmithKline has highlighted that a number of RMMs are being utilised, but their widespread adoption in the pharmaceutical industry faces several barriers including: economic and financial; institutional; legislative and regulatory; and technical. Another important area of pharmaceutical product analysis is that of oxidative stability testing. A comparison was undertaken of two machines for measuring oxidative stability (the Rancimat (Metrohm)) and the ACL Instrument (ACL Instruments, Switzerland) to investigate their performance and assess their applicability for the testing of pharmaceutical excipients. The testing of corn oil, selected as a model substance, revealed a strong correlation between the results from the two machines. An aspect of oxidative stability testing is concerned with residual levels of peroxides therefore the ACL Instrument was compared to iodometric titration, a highly empirical traditional approach, for the quantification of peroxides in corn and Menhaden oil. Good correlation was found between peroxide levels measured by the two methods, but the results from the ACL Instrument showed a dependence on oil type, meaning that a standard would be required each oil type. Additional testing was carried out on polysorbates, pharmaceutical excipients that are known to pose stability issues in final formulations, due to containing residual amounts of peroxides. Using the ACL Instrument it was possible to detect differences in oxidative stability between grades and types of polysorbate. It was not possible to discriminate between batches of the same grade. The final testing that is carried out on pharmaceutical products stored in glass vials, is container integrity testing. High voltage leak detection (HVLD) is one method utilised and concerns exist as to whether the high voltages present can cause ozone formation in the container headspace, leading to degradation of the drug product. A method involving potassium indigo trisulphonate was developed for the detection of ozone and a test protocol implemented. This approach showed that ozone was produced in containers with a low fill volume, but at a very low concentration and hence it should not affect the stability of the final product. The research carried out in this thesis has drawn together evidence on RMMs; furthered understanding of the applicability of oxidative stability testing for pharmaceutical excipients; and has highlighted the importance of investigating the effect of container integrity testing methods on the final pharmaceutical product.

Cyclin-dependent kinase 2 (CDK2) and Nek2 (Never-In-Mitosis A related kinases) are cell-cycle associated serine-threonine kinases that play an important role in the regulation of cellular proliferation and mitosis. Aberrant CDK2 and Nek2 activity are strongly associated with cancer, and inhibitors of these protein kinases are of potential therapeutic use as antitumour agents. Inhibitors of Nek2 –Previous studies identified a series of 2-aminoaryl-6-ethynylpurines (e.g. 42), as potent and selective irreversible Nek2 inhibitors with good antitumour activity in vitro and in vivo. 6-Ethynylpurine 42 binds within the ATP domain of Nek2 via a triplet of hydrogen bond interactions with the hinge region, enabling a covalent reaction between Cys-22 and the 6-ethynyl substituent. However, subsequent SAR studies indicated possible off-target activity for this series, which has been investigated through the synthesis and evaluation of control compounds engineered to be inactive against Nek2. With a view to abolishing Nek2-inhibitory activity without imposing dramatic structural changes, the effect of methylation at the purine N-7 (64) and N-9 (63) positions was studied. The corresponding isosteric 6-cyanopurine derivatives (65, 66, and 67) were also synthesised, which were expected to resemble closely 6-ethynylpurines without the capacity to react covalently. Evaluation of these derivatives in cell-based assays confirmed the presence of a growth-inhibitory activity unrelated to Nek2 inhibition. Regioselective N-7 methylation of 65 proved challenging, and a novel approach was developed whereby initial N-9 protection enabled selective methylation at the purine N-7 position, with concomitant loss of the N-9 protecting group giving the target purine 67. Kinetic studies have also been conducted with 42, 63 and 64 to assess the impact of N-7/N-9 methylation on the chemical reactivity of the 6-ethynyl ‘warhead’. A model system was developed to investigate a possible correlation between the chemical and biological reactivity of the 6-ethynyl group of the purine derivatives with thiols. Inhibitors of CDK2 –Previous studies have resulted in the identification of the purine CDK2 inhibitor 60, found to inhibit CDK2 in a time-dependent manner (IC50 = 63 nM) via conjugate addition of a lysine residue (Lys89), located in the ‘hinge region’ of the v ATP-binding domain, to the vinyl sulfone functionality of 60. This is thought to represent the first example of irreversible CDK2 inhibition, and prompted more detailed investigations with 60. Compound 60 exhibited a short half-life in both plasma (44 min) and medium (19 min), ascribed to hydration of the vinyl sulfone. Therefore, a priority was to improve the chemical stability of 60 without compromising inhibitory potency and time-dependent inhibition. Using the crystal structure of 60 in complex with CDK2 to guide inhibitor design, a range of derivatives (162-168) have been synthesised bearing α-substituents (R) on the vinyl sulfone group. In addition, synthetic methodology was developed for the synthesis of the corresponding 2-hydroxyalkyl products which are putative ATPcompetitive inhibitors. From the biological studies conducted on purine derivatives 162- 168, only the α-chlorovinyl sulfone compound 167 (IC50 (4 h) = 14 nM) has emerged as of particular interest, whereas others have shown competitive CDK2 inhibition, confirmed by X-ray crystallography. The β-position of the vinyl sulfone moiety has been briefly explored with purines (323, 333 and 339). In this series, one inhibitor (323) has been shown to bind covalently to CDK2 by structural biology studies. Alternative ‘warheads’ to the vinyl sulfone group have been investigated and exhibited competitive CDK2 inhibitory activity in a time-dependent inhibition assay. Finally, insertion of the vinyl sulfone warhead into known CDK2 inhibitors was investigated in preliminary studies.

Lyophilisation or freeze drying is the preferred dehydrating method for pharmaceuticals liable to thermal degradation. Most biologics are unstable in aqueous solution and may use freeze drying to prolong their shelf life. Lyophilisation is however expensive and has seen lots of work aimed at reducing cost. This thesis is motivated by the potential cost savings foreseen with the adoption of a cost efficient bulk drying approach for large and small molecules. Initial studies identified ideal formulations that adapted well to bulk drying and further powder handling requirements downstream in production. Low cost techniques were used to disrupt large dried cakes into powder while the effects of carrier agent concentration were investigated for powder flowability using standard pharmacopoeia methods. This revealed superiority of crystalline mannitol over amorphous sucrose matrices and established that the cohesive and very poor flow nature of freeze dried powders were potential barriers to success. Studies from powder characterisation showed increased powder densification was mainly responsible for significant improvements in flow behaviour and an initial bulking agent concentration of 10-15 %w/v was recommended. Further optimisation studies evaluated the effects of freezing rates and thermal treatment on powder flow behaviour. Slow cooling (0.2 °C/min) with a -25°C annealing hold (2hrs) provided adequate mechanical strength and densification at 0.5-1 M mannitol concentrations. Stable bulk powders require powder transfer into either final vials or intermediate storage closures. The targeted dosing of powder formulations using volumetric and gravimetric powder dispensing systems where evaluated using Immunoglobulin G (IgG), Lactate Dehydrogenase (LDH) and Beta Galactosidase models. Final protein content uniformity in dosed vials was assessed using activity and protein recovery assays to draw conclusions from deviations and pharmacopeia acceptance values. A correlation between very poor flowability (p<0.05), solute concentration, dosing time and accuracy was revealed. LDH and IgG lyophilised in 0.5 M and 1 M mannitol passed Pharmacopeia acceptance values criteria with 0.1-4 while formulations with micro collapse showed the best dose accuracy (0.32-0.4% deviation). Bulk mannitol content above 0.5 M provided no additional benefits to dosing accuracy or content uniformity of dosed units. This study identified considerations which included the type of protein, annealing, cake disruption process, physical form of the phases present, humidity control and recommended gravimetric transfer as optimal for dispensing powder. Dosing lyophilised powders from bulk was demonstrated as practical, time efficient, economical and met regulatory requirements in cases. Finally the use of a new non-destructive technique, X-ray microcomputer tomography (MCT), was explored for cake and particle characterisation. Studies demonstrated good correlation with traditional gas porosimetry (R2 = 0.93) and morphology studies using microscopy. Flow characterisation from sample sizes of less than 1 mL was demonstrated using three dimensional X-ray quantitative image analyses. A platinum-mannitol dispersion model used revealed a relationship between freezing rate, ice nucleation sites and variations in homogeneity within the top to bottom segments of a formulation.

Orally disintegrating tablets (ODTs) which are also referred to as orodispersible and fast disintegrating tablets, are solid oral dosage forms which upon placing on the tongue, disperse/disintegrate rapidly before being swallowed as a suspension or solution. ODTs are therefore easier and more convenient to administer than conventional tablets and are particularly beneficial for paediatric and geriatric patients, who generally have difficulty swallowing their medication. The work presented in this thesis involved the formulation and process development of ODTs, prepared using freeze-drying. Gelatin is one of the principal excipients used in the formulation of freeze-dried ODTs. One of the studies presented in this thesis investigated the potential modification of the properties of this excipient, in order to improve the performance of the tablets. As gelatin is derived from animal sources, a number of ethical issues surround its use as an excipient in pharmaceutical preparations. This was one of the motivations, Methocel™ and Kollicoat® IR were evaluated as binders as alternative materials to gelatin. Polyox™ was also evaluated as a binder together with its potential uses as a viscosity increasing and mucoadhesive agent to increase the retention of tablets in the mouth to encourage pre-gastric absorption of active pharmaceutical ingredients (APIs). The in vitro oral retention of freeze-dried ODT formulations was one property which was assessed in a design of experiments – factorial design study, which was carried out to further understand the role that formulation excipients have on the properties of the tablets. Finally, the novel approach of incorporating polymeric nanoparticles in freeze-dried ODTs was investigated, to study if the release profile of APIs could be modified, which could improve their therapeutic effect. The results from these studies demonstrated that the properties of gelatin-based formulations can be modified by adjusting pH and ionic strength. Adjustment of formulation pH has shown to significantly reduce tablet disintegration time. Evaluating Methocel™, in particular low viscosity grades, and Kollicoat® IR as binders has shown that these polymers can form tablets of satisfactory hardness and disintegration time. Investigating Polyox™ as an excipient in freeze-dried ODT formulations revealed that low viscosity grades appear suitable as binders whilst higher viscosity grades could potentially be utilised as viscosity increasing and mucoadhesive agents. The design of experiments – factorial design study revealed the influence of individual excipients in a formulation mix on resultant tablet properties and in vitro oral retention of APIs. Novel methods have been developed, which allows the incorporation of polymeric nanoparticles in situ in freeze-dried ODT formulations, which allows the modification of the release profile of APIs.

Angiotensin converting enzyme (ACE) inhibitors lisinopril and ramipril were selected from EMA/480197/2010 and the potassium-sparing diuretic spironolactone was selected from the NHS specials list for November 2011 drug tariff with the view to produce oral liquid formulations providing dosage forms targeting paediatrics. Lisinopril, ramipril and spironolactone were chosen for their interaction with transporter proteins in the small intestine. Formulation limitations such as poor solubility or pH sensitivity needed consideration. Lisinopril was formulated without extensive development as drug and excipients were water soluble. Ramipril and spironolactone are both insoluble in water and strategies combating this were employed. Ramipril was successfully solubilised using low concentrations of acetic acid in a co-solvent system and also via complexation with hydroxypropyl-β-cyclodextrin. A ramipril suspension was produced to take formulation development in a third direction. Spironolactone dosages were too high for solubilisation techniques to be effective so suspensions were developed. A buffer controlled pH for the sensitive drug whilst a precisely balanced surfactant and suspending agent mix provided excellent physical stability. Characterisation, stability profiling and permeability assessment were performed following formulation development. The formulation process highlighted current shortcomings in techniques for taste assessment of pharmaceutical preparations resulting in early stage research into a novel in vitro cell based assay. The formulations developed in the initial phase of the research were used as model formulations investigating microarray application in an in vitro-in vivo correlation for carrier mediated drug absorption. Caco-2 cells were assessed following transport studies for changes in genetic expression of the ATP-binding cassette and solute carrier transporter superfamilies. Findings of which were compared to in vitro and in vivo permeability findings. It was not possible to ascertain a correlation between in vivo drug absorption and the expression of individual genes or even gene families, however there was a correlation (R2 = 0.9934) between the total number of genes with significantly changed expression levels and the predicted human absorption.

ODTs have emerged as a novel oral dosage form with a potential to deliver a wide range of drug candidates to paediatric and geriatric patients. Compression of excipients offers a costeffective and translatable methodology for the manufacture of ODTs. Though, technical challenges prevail such as difficulty to achieve suitable tablet mechanical strength while ensuring rapid disintegration in the mouth, poor compressibility of preferred ODT diluent Dmannitol, and limited use for modified drug-release. The work investigates excipients’ functionality in ODTs and proposes new methodologies for enhancing material characteristics via process and particle engineering. It also aims to expand ODT applications for modified drug-release. Preformulation and formulation studies employed a plethora of techniques/tests including AFM, SEM, DSC, XRD, TGA, HSM, FTIR, hardness, disintegration time, friability, stress/strain and Heckel analysis. Tableting of D-mannitol and cellulosic excipients utilised various compression forces, material concentrations and grades. Engineered D-mannitol particles were made by spray drying mannitol with pore former NH4HCO3. Coated microparticles of model API omeprazole were prepared using water-based film forming polymers. The results of nanoscopic investigations elucidated the compression profiles of ODT excipients. Strong densification of MCC (Py is 625 MPa) occurs due to conglomeration of physicomechanical factors whereas D-mannitol fragments under pressure leading to poor compacts. Addition of cellulosic excipients (L-HPC and HPMC) and granular mannitol to powder mannitol was required to mechanically strengthen the dosage form (hardness >60 N, friability <1%) and to maintain rapid disintegration (<30 sec). Similarly, functionality was integrated into D-mannitol by fabrication of porous, yet, resilient particles which resulted in upto 150% increase in the hardness of compacts. The formulated particles provided resistance to fracture under pressure due to inherent elasticity while promoted tablet disintegration (50-77% reduction in disintegration time) due to porous nature. Additionally, coated microparticles provided an ODT-appropriate modified-release coating strategy by preventing drug (omeprazole) release.