If they are to enter routine clinical application, new radiopharmaceuticals must have rapid and simple labelling procedures, ideally inserting the radionuclide at the last synthetic step with no need for further purification. This thesis describes the development of new targeted strategies for molecular imaging. Targeted nanoparticles have great potential for application as radionuclide molecular imaging agents but are subject to several limitations, including complex radiolabelling procedures, slow pharmacokinetics, low uptake in target tissue, and potential toxicity. We have discussed a targeted nanoparticle system comprising biocompatible materials with intrinsic affinity for readily-prepared radiotracers such as 18F-fluoride and 99mTc-technetium bisphosphonate derivatives. Such a system would offer simple labelling, and signal amplification (each particle can deliver many radionuclides). To overcome slow pharmacokinetics we proposed to exploit pretargeting, whereby the radionuclide-nanoparticle bond can form in vivo. Firstly, a large pretargeting agent (nanoparticle) that has affinity for both its target tissue and radionuclide probe, accumulates at the target site slowly over a period of time. This is followed by a chasing step, where a small radionuclide probe (18F-fluoride and 99mTc-bisphosphonate) distributes rapidly to the pretargeting agent, while untargeted circulating radionuclide probe clears rapidly from the blood pool. The reader is first introduced to the topics of radiopharmaceutical particulates and pretargeting, emphasing the requirement for novel radiopharmaceutical targeting methods. A literature review discussing fluoride affine materials was performed to guide initial screening experiments. We screened many inorganic nanoparticulate materials for binding to 18F-fluoride. Of the materials tested, hydroxyapatite (HA) and Alhydrogel showed the most efficient binding to 18F-fluoride. The 18F-HA interaction was highly stable in serum, while the 18F-Alhydrogel interaction was moderately stable in serum. HA materials were prepared via wet chemical precipitation. The effect of synthesis termperature and post-synthesis treatment was investigated. Stabilisation and functionalisation of HA nanoparticles with various ligands was discussed. Synthesis temperature did not greatly affect particle properties, while calcination and hydrothermal post-synthesis treatments controlled particle morphology and crystallinity. HA particles formed stable colloidal solutions when functionalised with sodium hexametaphosphate (SHMP) and polyethyleneglycol-bisphosphonates (PEG-BP). The bisphosphonate -Alendronate was used to link small molecules such as amino acids and fluorescein isothiocyanate to HA surfaces, for potential targeting applications. Porous hollow silica particles were prepared using a novel templating method, using HA as core material. However, 18F-fluoride showed poor affinity for these materials. A novel bifunctional bisphosphonate chelator, A//A/-bis(quinoylmethyl)pamidronate-amine (BQMPA) was prepared and its potential as a probe for SPECT and fluorescence imaging was investigated. A DOTA like bisphosphonate - BPAMD (a literature compound) - and its novel MCu radiolabelled complex were prepared. The preparation of 99mTc and Re-BQMPA complexes resulted in the formation of multiple products. The Cu-BPAMD complex was identified as a single complex and was kinetically stable in serum over 24 h. Both 99mTc-BQMPA and 64Cu-BPAMD showed high affinity for HA materials. We screened inorganic nanoparticulate materials for binding to 99mTc- and 64Cu-bisphosphonates. We identified that 99mTc(CO)3-DPA-Ale (99mTc labelled dipicolylamine-alendronate) and Cu-BPAMD bind to a wide range of metal oxide materials with high efficiency. HA and Alhydrogel were chosen as lead materials for further in vitro and in vivo investigations.

The work in this thesis describes the cloning, expression purification and characterisation of human CypA and FKBP12 proteins. Various biophysical techniques were used to study and characterise the interactions of different ligands with these target proteins. Vectors containing the genes encoding each of these, for expression of both wild-type protein and protein with a hexa-histidine tag, have been cloned for both hCypA and hFKBP12. Crystals of hCypA have been grown, and the structures, in complex with a range of small molecules including dipeptides, and dimedone derived ligands, have been solved. hCypA crystal complexes were also used to further explore the nature of the ‘crystal dissociation constant, Kcryst’ using the depetide ligand Val-Pro. Crystals were soaked in various ligand concentrations and by solving the X-ray structures of these complexes, ligand occupancies could be determined providing an experimental value for the equilibrium binding constant, Kcryst = 23mM. This compares well with a Ki value 19mM measured using an enzyme inhibition assay. Protein-ligand binding data measured using a range of methods is also presented, including fluorescence spectroscopy, mass spectrometry and surface plasmon resonance. It has also been demonstrated how the technique of surface plasmon resonance may be optimised to provide a sensitive ligand-binding assay. By tethering protein to a Ni2+ chip via an N-terminal hexa-his tag followed by covalent linking via primary amines, a stable and active surface was obtained. This allowed a competition binding assay to be developed, which allows the binding of ligands of Mr 250-500Da to be measured. The work done here should help further the design of potential inhibitors of hCypA, and in the measurement of their binding affinities; and possibly aid in the future design of anti HIV-1 therapies.

Elastase is a serine protease that has been implicated in a number of inflammatory conditions including rheumatoid arthritis and cystic fibrosis. These conditions are thought to result from an increased amount of active elastase in the body, caused by insufficient inhibition by endogenous inhibitors. Elastase has the ability to degrade many tissue components and this excessive tissue damage causes the onset of a variety of conditions. This knowledge has led to an increased interest in the production of elastase inhibitors with the hope of developing an inhibitor, which can be of therapeutic use <i>in vivo. </i>As a result we have synthesised a series of novel β-lactams since β-lactam compounds are known to be mechanism-based inhibitors of serine proteases. Electrospray ionisation mass spectrometry (ESI-MS) identified a novel mode of inhibition, for β-lactams which possess a hydroxyl group present at the C3 position, resulting in the formation of an acyl-enzyme intermediate which was found to be extremely stable. Enzyme kinetic studies demonstrated that the β-lactams had k_ass values ranging from 1000 - 10,000 M^-1 s^-1. The novel β-lactams were also assayed for activity against trypsin. ESI-MS confirmed that they were also inhibitors or trypsin but enzyme kinetic studies revealed that they were more active towards elastase. Therefore the novel β-lactams were found to be stable, potent and specific inhibitors of elastase that may of therapeutic use in the treatment of many inflammatory conditions.

Microneedle mediated drug delivery is an amalgam of the conventional transdermal patch and the hypodermic needle injection. It offers an improved drug delivery technique without the limitations of the above methods. The ability of microneedles to increase permeability of substances in the skin has been established in the literature. However, a quantitative method for predicting the performance of microneedle devices prior to their fabrication is yet to be fully developed. The contribution of this research is a theoretical framework for modelling and optimizing microneedle array design to obtain desired drug delivery rate while taking into account the transport and mechanical properties of the skin. This is achieved by exploring various theories surrounding transdermal drug transport. The existing theories are then used to develop models to link the microneedle array design parameters with drug transport properties such as permeability and drug concentration in blood. Numerical simulations and theoretical analyses that are carried out in this PhD research indicate that microneedle design has a significant effect on drug delivery. An algorithm was developed for solving the series of equations presented, thus obtaining a framework which is applied to predict performance of microneedle arrays in vivo. Some practical scenarios are also simulated to demonstrate the applicability of the developed framework. For example, numerical simulations of transdermal delivery of Fentanyl show that varying the design parameters such as penetrated length of microneedle and the tip radius of microneedles affected the peak blood concentration. Similarly, the developed framework was used to obtain the optimum microneedle design to calculate the desired peak blood concentration similar to that obtained using conventional patch system. This study is relevant as it provides a better understanding of microneedle mediated drug delivery process and it orchestrates the design and hence, fabrication of more efficient microneedle based drug delivery devices.

The ability to predict accurately the dermal permeation of a chemical is important for the development of new therapeutic formulations in transdermal drug delivery and also for the assessment of the potential risks of environmental chemicals and agents used in cosmetics industry. There have been a large number of studies investigating skin permeability, both experimentally- and theoretically-based. The experimental measurement of skin permeability is a time-consuming and difficult process and, when put in the context of the great numbers of chemicals that may come into contact with the skin, it is also costly. As a consequence, there has been a great interest over the past years to 'predict' the ability of a compound to cross the skin using its physicochemical characteristics and a number of mathematical models have been reported. Most models are based on experimental data, with skin permeability linearly correlated to the physicochemical properties and/or molecular structure parameters of the chemical compounds. A multilinear regression method is often used to fit available experimental data and such empirical models are referred to as quantitative structure-property relationships (QSPRs). Many QSPR models relate skin permeability to the physicochemical properties of the solutes while others use molecular structure properties. Successful application of such models can realise a marked reduction in the number of potentially therapeutic molecules requiring synthesis and validation since they can be precluded from study on the basis of their predicted lack of skin permeability. In terms of skin permeability data with respect to QSPR analysis, the science has not been developed significanijy since the publication of what is now universally known as the Flynn dataset (Flynn 1990). The most widely cited model that was developed on the basis of the Flynn dataset was the Potts and Guy (1992) model and since then a number of alternative models have been developed using the Flynn and other subsequendy assembled datasets. The aim of this study was to determine the limitations of the existing models and to address any deficiencies by the development of new mathematical models. Databases containing measured and well-defined skin absorption data are a key first step in the development of QSPR models, which might improve the understanding of the dermal absorption process for chemicals. Therefore the first objective was to develop a more stringently controlled database that induded parameters derived under more strictly defined experimental conditions. These data could then enable the current models to be re-evaluated and accordingly a detailed analysis of all the available in vitro dermal absorption data to be conducted with a view to classifying the data according to the corresponding experimental conditions employed so as to produce a number of data subsets. Subsequendy, advanced computational regression modelling methods, such as the Gaussian method, were applied to develop the most efficient model in terms of statistical fit. The Gaussian process (GP) has not been applied previously to skin permeability data. The potential of induding additional descriptors such as molecular weight, lipophilicity, Fedors' solubility parameter, hydrogen bond acceptor and donor capacity into any developed equations with a view to improving accuracy was investigated. For comparison reasons, a new linear regression model was also developed based on the above-mentioned 5 additional descriptors (5f linear regression model). The GP model yielded a predictive model that provided a significandy more accurate estimate of skin absorption than previous models across a wider range of molecular properties. It proved to be particularly capable of providing excellent predictions where previous studies have shown QSPRs to fail: at high log P and MW of penetrants. The results indicate that, in terms of statistical performance, the Gaussian model was better than the 5f model. This suggested that, statistically, a nonlinear approach, as employed herein, is more appropriate for analysis than linear techniques. The relative accuracy of the GP, 5f linear regression and Potts and Guy (1992) models were also compared. Permeation data were obtained under carefully controlled conditions and these were correlated to values calculated from the application of various models. Apart from comparing the experimental values with those predicted from each model, all deviations were recorded and the performance of each model was assessed. The flux values of two drug candidates, ibuprofen and furosemide, determined using the GP and 5f models, agreed well with those obtained experimentally. However, procaine hydrochloride diffused at a rate that was slower than that predicted by all models and paracetamol diffused at a much higher rate than was predicted from the GP model. The latter results could possibly be due to the relatively small degree of paracetamol ionisation. As a resutt, the effect of ionisation on permeability was measured by determining the flux of a single ionisable drug at three different pH values. The experimental results obtained were compared with the linear ionisation model, in which log D values were incorporated into the equation instead of log P values. It was concluded that ionisation, together with nonlinearity, were some of the most important factors that require further consideration when designing new models to predict dermal absorption. A series of more strictly defined databases was successfully assembled during the course of this study and used to propose novel computational models for dermal absorption. Such models showed satisfactory predictive capacity but do require further development, particularly in the case of ionisable compounds. However, the development of such databases alone is of great use for future model development.

Wet bead milling, in which the drug is milled in presence of stabilisers such as polymers and surfactants, has enabled the formulation of poorly water-soluble drugs as nanoparticles, with five products having reached the market. During the milling process, the polymer and/or surfactant adsorbs onto the freshly cleaved drug surfaces to provide ionic or steric stabilisation. Despite the success of wet bead milling, mastery of the mechanism behind nanoparticle stabilization is still lacking. To investigate whether any relationship exists between drug, stabiliser and stabilisation, eight structurally different poorly water-soluble drugs were milled in presence of thirteen different pharmaceutically acceptable stabilisers and the resultant particle size determined by photon correlation spectroscopy. Nanoparticles of the BCS class II drugy griseofulvin, could only be produced in presence of anionic stabilisers namely sodium dodecyl sulphate, aerosol-OT or hydroxypropylmethylcellulose acetate succinate. Surfactant adsorption isotherms obtained indirectly by measuring their depletion from solution revealed a maximum surfactant adsorption of ~ 2.2 mg/m2 on the griseofulvin nanoparticle surfaces. The use of ionic surfactants/polymers in oral formulations is however sub-optimal. Consequently, polymer-surfactant co-stabilisation, used to take advantage of the synergy between ionic and non-ionic stabilisers, was investigated by the inclusion of the non-ionic polymer hydroxypropylmethylcellulose (HPMC) into the anionic surfactant-drug slurry prior to milling. The effect of varying HPMC molecular weight and concentration on griseofulvin nanoparticle production was established. Polymer adsorption isotherms were obtained directly via small angle neutron scattering.

The adult stage of the parasitic nematode <i>Brugia malayi</i> can live in the host lymphatics for many years and must have an extremely effective immuno-suppressive mechanism that prevents rejection. We have discovered that this parasite can induce alternatively activated IL-4 dependent macrophages that can block proliferation of other cells via a receptor-mediated mechanism. The proliferative block is reversible and is not a result of apoptosis. Furthermore, these suppressive cells can reduce the proliferation of a wide range of human cancer cell lines. These data demonstrates that <i>B. malayi</i> can lead to the activation of a novel mechanism of proliferative suppression, via IL-4 dependent macrophages. These macrophages may have important roles in altering host immune responses during parasitic infection and may even have the potential to reduce tumour cell growth. Another feature of <i>B. malayi </i>infection is a bias towards a type 2 immune response. To investigate the role that the <i>B. malayi </i>recruited antigen presenting cells have on naive T cells, the suppressive macrophages recruited by <i>B. malayi </i>was used to stimulate naive T cells from TCR transgenic mice. Although the naive T cells were inhibited by parasite-induced macrophages during primary stimulation, they proliferated normally upon secondary stimulation and were not rendered anergic. However, naive T cells primed by suppressive macrophages differentiated into IL-4 producing Th2 cells upon secondary stimulation instead of IFN-g producing Th1 cells, as has been previously described. Th2 differentiation was associated with the inhibition of (or failure or stimulate) IFN-g production during primary stimulation. Interestingly, blocking antibodies against TGF-b (but not IL-10) restored the differentiation of IFN-g producing Th1 cells. These data indicate that T cells exposed to parasite induced alternatively activated macrophages are driven towards Th2 differentiation. This may be an important factor in the Th2 bias that accompanies helminth infection.

Introduction: Medicines are used with the intention of benefitting from their effect. The effects of medicines can also be undesirable and potentially lead to harm. A drug-related problem (DRP) is a term used to describe problem(s) that exist in the use of medicines. There remains a distinct paucity of data on the epidemiology of DRPs in children with kidney disease. Aim: To investigate the epidemiology of DRPs in children with kidney disease in clinical practice at tertiary Paediatric Nephrology units. Methods: Study 1: Prospective observational study on the characteristics of DRPs in hospitalised children with kidney disease. Study 2: Randomised control trial on clinical pharmacist (CP) interventions in resolving DRPs on the renal outpatient clinic. Results: Study 1: A total of 127 patients were recruited and a total of 203 DRPs were identified. The incidence of DRP was 51.2% (95% CI 43.2-60.6%) of patients reviewed by the CPs. The number of medicines prescribed per child was the only significant risk factor for the occurrence of DRPs (OR 1.06, 95% CI 1.02-1.10, p=0.002). The majority of DRPs were minor in clinical significance (68%, n=138/203). The predominant DRPs were sub-optimal drug effect. These DRPs were associated with drug selections and dosage errors. Study 2: A total of 100 patients were recruited (Control n=53, Intervention n=47). The trial showed no effect of intervention in the resolution of active DRPs (p=0.96) between the Control and Intervention arms. Conclusion: DRPs are common in children with kidney disease and necessitate a comprehensive approach to their identification and resolution. Their characteristics in both settings are different even though the majority of them shared a similar level of clinical significance. Further research is required to evaluate the effectiveness of pharmacists’ intervention in resolving DRPs at the outpatient clinics.

Zinc (ionic form Zn2+) is a common trace element in the forebrain, and is especially enriched in the hippocampus, a brain structure important for learning and memory. A large amount of vesicular Zn2+ which is thought to be released upon presynaptic depolarisation is found at synapses formed by the axons of dentate granule cells (GCs), known as mossy fibres (MFs). Zn2+ inhibits NMDA and GABAA receptors (NMDAR and GABAAR) at mono-synaptic inputs between MFs and CA3 pyramidal neurons but its role in synaptic integration in the dentate gyrus remains elusive. Whole-cell recordings were obtained from GCs held in voltage-clamp in acute rat hippocampal slices. One tungsten electrode was positioned in stratum lucidum (SL) of CA3b to activate MFs and another in stratum granulosum (SG) to directly stimulate dentate interneurons. Evoked synaptic currents were blocked by superfusion of the GABAAR antagonist bicuculline implying that they were mediated by GABAARs. In contrast, the AMPA/kainate receptor antagonist NBQX abolished SL evoked inhibitory postsynaptic currents (IPSCs) but had little effect on IPSCs evoked by SG stimulation. Similarly, the group 2 metabotropic receptor agonist DCG-IV depressed SL but not SG evoked IPSCs. These results imply a poly-synaptic inhibitory feedback projection from CA3 to the dentate gyrus via recurrent MFs, and a mono-synaptic input from dentate interneurons to GCs. Zn2+ reversibly depressed evoked IPSCs whereas superfusion of different Zn2+ chelators had the opposite enhancing effect. Blocking T-type Ca2+ channels abolished the effect of Zn2+ chelators. When recording from dentate basket cells, Zn2+ chelation increased spike width, decreased spike threshold, enhanced NMDAR-mediated excitatory postsynaptic currents (EPSCs) and facilitated T–type Ca2+ currents. Finally, chelation of Zn2+ narrowed the time window for integration of perforant path inputs and facilitated GC spiking. Together, the results demonstrate that Zn2+ modulates MF–interneuron–GC communication and thus regulates information transfer to dentate and hippocampal networks.

Pharmaceutical compounding corresponds to the preparation of customised medicines in order to meet the specific needs of patients, which cannot be met by the proprietary medicines provided by the pharmaceutical industry. Historically, pharmaceutical compounding dates back to the very origins of pharmacy and, ever since, it has been an integral part of pharmacy practice. Nevertheless, little is known regarding current compounding practices in Europe and, therefore, the aim of this project was to identify and characterise the oral compounded medicines most frequently dispensed in European hospital pharmacies. The research method adopted was a large-scale, international (European) survey and the research instrument was a self-completion (country-specific) questionnaire. A total of 11 European countries were included in the research: Portugal, UK, Switzerland, Poland, Netherlands, Denmark, Slovenia, Finland, Spain, France and Germany. For most countries, a purposive sample of hospitals was contacted and invited to contribute data regarding the oral compounded medicines most frequently dispensed in their pharmacies. The pilot-study was launched in Portugal but fieldwork was undertaken in most countries. Information regarding legislation, professional organisations and information sources relevant to pharmaceutical compounding was also collected. The oral compounded medicines most frequently dispensed in hospital pharmacy varied considerably throughout Europe, from traditional cachets in Poland to complex tablets in the Netherlands and Denmark. A wide range of active substances, including NTI drugs, and dosage strengths were dispensed. Compounded medicines were prepared individually and also in batches of variable sizes. There is little consistency of compounding practices in Europe and there is a need for common legislation, professional organisations and information sources. This project corresponds to the largest and most complex research in pharmaceutical compounding across Europe and aims to contribute to the harmonisation of quality and safety of compounded medicines in Europe.