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A high-quality chemical probe should possess the qualities of being highly specific, potent, and selective against off-target interactions, in order to enable target validation. By contrast, low-quality compounds, which display properties of promiscuity, reactivity and instability can in turn be misleading: such compounds, referred to as Pan-Assay Interference Compounds (PAINS) need to be removed from screening libraries. Target engagement and proteomic studies can often detect undesirable physicochemical properties or polypharmacology within small molecules, as well as help annotate and clean screening libraries and prevent compounds entering biology projects without precautions. Herein are presented a number of target engagement techniques used to annotate compounds. The introduction summarises currently available techniques and provides a literature review pertaining to the Cellular Thermal Shift Assay (CETSA). The second chapter describes how the use of chemoproteomic techniques, including CETSA, has led to the discovery of a number of undesirable characteristics of a literature compound SJ-172550. The third chapter delves into mass spectrometry (MS)-based CETSA techniques, and describes the development of an algorithm to analyse MS-CETSA output data for target validation, using both open-access datasets as well as the dataset obtained from the analysis of a known protein arginine methyltransferase 5 (PRMT5) inhibitor. The fourth chapter describes the use of Immunoblot-CETSA to investigate cellular target engagement of peroxisome proliferator-activated receptor γ (PPARγ)-targeting compounds in the context of downstream induction of the uncoupling protein, UCP1. Subsequently, the fifth chapter concerns the development of an AlphaScreen-based target engagement assay with a high-throughput capacity, and the necessary optimisation steps, and provides the link between AlphaScreen-CETSA and MS-CETSA. The sixth chapter pertains to the proteomic analysis of PPARγ-expressing cells, including an MS-CETSA experiment involving a known PPARγ agonist, rosiglitazone, to investigate its off-target interactions. Chapters 5 and 6 together point to the combined use of AlphaScreen-CETSA for hit detection and MS-CETSA for hit follow-up.

Novel steroidal oestrogens, that incorporate some of the structural motifs of non-steroidal antioestrogens, were synthesised and tested for their actions on the BK channel. These novel compounds were first checked for purity and structure using, where appropriate, 1H NMR, 13C NMR, IR spectroscopy, mass spectroscopy, thin layer chromatography, melting point analysis, elemental analysis and X-ray crystallography. Using Oestrone as the starting compound, the following five novel compounds were synthesised; DME-Oestrone, Quat-DME-Oestrone, DME-Oestradiol, Quat-DME-Oestradiol and Oestrone-Oxime. Two of the derivatives, Quat-DME-Oestrone and Quat-DME-Oestradiol, incorporated a quaternary ammonium side-chain making them membrane impermeable.

Nanomaterials are generally defined as chemical substances or materials that contain particles with one or more dimensions less tl1an 100 nanometres in size. They may be either engineered or naturally occurring, but have unique properties due to a vastly increased surface area to volume ratio when compared to non-nano (bulk) materials. This provides the potential for the development of a wide range of enhanced formulations with superior efficacy including applications in oral health care .. As the properties of a material change at the nano-scale, there are concerns that the toxicological profile of these materials may also change. Size is only one factor; changes in shape, surface chemistry, chemical composition, porosity and solubility all contribute to the overall biological toxicity profile of a nano-scale ingredient. Established links between the specific properties of a nanomaterial and toxicity are not well understood, leaving an important data gap in the literature. The purpose of this work was to utilise in vitro oral epithelial models for the assessment of safety profiles of nanomaterials for applications in next generation oral care products. Four commercially sourced nanomaterials were analysed, alongside respective bulk counterparts already found within oral care product formulations. These nanomaterials comprised of two nano-zinc oxides (ZnO), silicon dioxide (SiOz), titanium dioxide (TiOz) and hydroxyapatite (Cas(OH)(P04)'). Comprehensive characterisation of each material was carried out using a range of analytical techniques to identify any structure-function relationships in vitro. Initial toxicity screening experiments were conducted using a non-keratinised oral epithelial cell monolayer (H376 cell line) with both cell viability and lysis analysed using MTT and LDH assays respectively. Materials were investigated further using two 3-dimensional tissue models representative of the main tissue types constituting the human oral mucosa: non-keratinised buccal (RHO) and keratinised gingival (GIN-lOO) models. Nanomaterial uptake in the models was investigated using confocal microscopy with a styrl dye (FM 1-43). This led to the development of a novel, high throughput fluorescent assay as a potential method for screening nanoparticle-uptake. Results highlighted the complexities involved with nano-characterisation in biological media using current techniques. A wide variety of particle shapes and sizes were recorded between different nanomaterials, with results being dependent upon the sample preparation steps and specific methods of analyes used. These disparities represent the current challenges experienced by both researchers and regulators of nanotechnology at the present time. ZnO \vas observed to be the most cytotoxic material during monolayer screening, at concentrations exceeding 0.3125% w/v when delivered in protein-free media. Differences between bulk and nanomaterial properties were recorded for all the materials, except for Ti02, but these did not necessarily transfer to effects seen in the more representative 3-D models. Cytotoxicity results from both R1--10 and GIN-lOO models exemplified the disparity between sensitivity of monolayer and the natural stratified tissue structure of human oral mucosa. Keratinised gingival tissue models showed slgnificantly greater durability over the less robust buccal model, in both cytotoxicity assays and IL- lcx cytokine response. Of all materials examined, cellular uptake was only observed for nano-Si02. This was the only material detected trafficking inside the cell using the FM 1-43 styryl dye assay, with confocal data serving to verify the analysis of nanoparticle Internalisation using fluorescence. In conclusion, nanomaterials pose considerable difficulties during formulation and analysis in health care products. The risk of potential uptake and bioaccumulation or translocation to particularly sensitive areas of the body also requires further investigation. Nanomaterials have to be assessed on a case by case basis, and robust/consistent regulatory strategies developed to enable industry to produce and market novel but safe nanoparticle containing formulations. Risks to human health may be less of a hazard when applied to fully functioning healthy human tissue, especially in comparison to existing bulk material effects and current, accepted irritant ingredients (e.g. Sodium lauryl sulphate).

Background: Pharmacovigilance (PV) is the science and activities involved in monitoring and developing the safety profile of all marketed medicines. Adverse drug reactions (ADRs) for medicinal products can be identified through postmarketing studies by methods of signal detection. Traditional, qualitative methods involve clinical review of cases, and coupled with modem, quantitative methods which have evolved as PV has grown, may help surveillance of the large number of medicinal products on the market today. This research aimed to investigate combining traditional and modern methods of signal detection by adding statistical weighting to adverse event telms identified as requiring further monitoring pre-marketing, to improve identification and evaluation of ADRs post -marketing. Methods: Four anti-diabetic drugs currently marketed were chosen to model the concept: gliclazide, pioglitazone, rosiglitazone and vildagllptin. Review of pre-marketing information for safety concerns highlighted two medical concepts: cardiac failure and acute pancreatitis. The Delphi method was adapted to identify and pliolitise terms for these concepts to add statistical weighting to. The weightings were applied to two datasets, both from the UK Yellow Card. Scheme (YCS): a two-year dataset (2005-2007) and a ten-year dataset (2000- 2010).

Paroxetine, a selective serotonin reuptake inhibitor, is a potent drug used for the treatment of depression for more than 20 years. Although numerous syntheses have been reported for this molecule, the manufacturing process still uses a resolution step, as it remains the shortest way to access paroxetine on a large scale. Our synthesis of paroxetine offers an alternative solution, delivering the final compound in only six steps from commercially available material. This has been possible thanks to the use of both organo- and organometallic catalysis to introduce the two chiral centres. Formaldehyde has been used in a direct proline-catalysed aldol reaction, allowing the selective introduction of a hydroxymethyl group with good atom economy. Optimisation of a cobalt-catalysed Kumada-type Csp3-Csp2 cross coupling reaction on a secondary bromide enabled us to introduce the aryl substituent in the 4-position. Furthermore, the generation of a configurationally labile cobalt intermediate in the cross coupling reaction has been successfully exploited to develop a diastereoselective arylation. The use of methanol for the introduction of a hydroxymethyl group via transfer hydrogenative coupling has also been briefly investigated for incorporation in an asymmetric synthesis of abacavir.

The use of proteins as therapeutics is one of the fastest growing sectors of the pharmaceutical industry, particularly monoclonal antibodies. However, a significant challenge in the development of such protein-based medicines is to counter aggregation of the proteins in solution (as these drugs are typically administered by injection). In solution form, aggregation of the normally monomeric protein ingredient affects therapeutic efficiency and reduces shelf life. Moreover, the rapid formation of aggregates in patients during the administration of therapeutic proteins can lead to immunological reactions which could be fatal. Hence, the long term storage of proteins in solution is discouraged. Lyophilization (vacuum drying) is considered to be an effective route for ensuring longer shelf life and better stability of protein therapeutics. However, aggregation can still occur, because the driving forces for aggregation (covalent as well as non-covalent interactions such as hydrogen bonds, van der Waals forces and hydrophobic interactions) are influenced by lyophilization induced changes in the pH, temperature, exposure to interfaces, and dehydration stress. Lyoprotectants such as sugars can counter the undesirable consequences of lyophilization depending upon their nature and potential. Several mechanisms have been proposed for the role of the lyoprotectants. The comprehensive investigation into the inherent nature and influence of lyoprotectants on a protein therapeutic during lyophilization is hence important. In this project an attempt has been made to develop a novel nanoscale screening methodology for the detection, quantification, characterization and prevention of protein aggregation. Initially, ferritin and then a polyclonal IgG (antiglucose-6-phospate dehydrogenase antibody) antibody have been used as model proteins for these studies. The effect of lyophilization on the level of aggregation of IgG was studied and compared to reports in the literature. IgG was exposed to seven cycles of lyophilization, where each cycle of lyophilization was followed by reconstitution and characterization. IgG was also lyophilized with different excipients (sucrose and mannitol, alone and in combination) in different molar ratios. In the liquid state, the formulations were characterized on the basis of particle size and antigen binding activity, whereas in the dry powdered form, the formulations were characterized by studying morphology, thermal stability, and secondary structural alterations in order to establish a relationship amongst the indicated properties. Atomic force microscopy (AFM), dynamic light scattering (DLS) and single particle tracking (Nanosight) were used to study particle size. The identification of different components at the nanoscale and general morphology were screened by AFM and scanning electron microscopy (SEM). Subsequently, the thermal properties and structural alterations respectively were analysed by differential scanning calorimetry (DSC) and infra read spectroscopy (ATR-FTIR) spectroscopy. The antigen binding activity was investigated by performing an indirect ELISA assay on the lyophilized formulations. Lyophilization of ferritin and IgG caused a significant decrease in the proportion of monomeric species was confirmed by AFM, DLS and Nanosight. Dimeric, lower-multimeric and larger aggregates existed in variable proportions for both ferritin and IgG. Powdered lyophilized ferritin formulations showed aggregation, increased crystallinity (concomitant decrease in amorphicity), porosity and flakiness which in case of IgG increased with repeated lyophilization. A consistent increase in the extent of aggregation (unfolding of Fabs and Fc) was detected by DSC and an increase in the beta-sheet structure coupled with structural re-arrangement within the components by ATR-FfIR. The presence of sucrose in IgG formulations resulted in reduced aggregation and enhanced porosity. The inclusion of Mannitol promoted crystallinity, decreased porosity when used alone, however, improved the efficiency of sucrose in combined formulations. The nature of crystals formed by mannitol during lyophilization was shown by SEM and confirmed by AFM. The data obtained from DLS, NTA, AFM, DSC,ATR, and SEM was consistent with by ELISA results which indicated a significant fall in IgG activity upon repeated lyophilization, and improvement in the activity when IgG was formulated with sucrose, which significantly enhanced in combination with mannitol. The benchmark provided by this work would serve as a precursor for developing a novel screening standard for optimizing and improving the therapeutic efficiency of other proteins besides furnishing a detailed account of the disparity in correlating the data from multiple novel techniques. The findings of our work can be directly translated to biotech and biopharm industries for the enhancement of protein based therapeutics.

Roll compaction is commonly used as a dry granulation technique in the pharmaceutical industry to produce tablets for formulations sensitive to heat and moisture. This thesis reports systematic studies on the behavior of pharmaceutical excipients in associated unit operations (i.e. roll compaction, milling, tabletting), as well as their correlations. Roll compaction experiments were carried out using an instrumented roll compactor with a gravity feeding system. The influence of the process parameters, material properties and powder conditioning were investigated Ribbons produced in roll compaction were granulated using an oscillating mill to investigate the milling process. A first order kinetics equation was introduced to describe the mass throughput of the granules. Using positron emission particle tracking technique, which provided a measurement of instantaneous velocity and the location of the ribbons, two milling regions (i.e. impact and abrasion) involving distinct fracture mechanisms were identified. Tabletting of the granules was performed using a universal test machine. A reduction in the compressibility and compactibility of the granules compared to the feed powders, due to work hardening, was also observed. A method was introduced to determine the optimized process conditions for roll compaction and milling through a close examination of the correlation between the unit operations.

An artificial liposome membrane system has been employed for in vitro screening of the human absorption of biologically active molecules for applications in nutrition and drug treatments. Initial work with molecules having small permeabilities demonstrated that they could not be measured using the technique since they were absorbed by the membrane. A critical innovation was to pre-treat the membrane by equilibrating it with the molecule of interest since this avoids the absorption problem but required more complex data analysis. Bioactive molecules with strong antioxidant and anti-cancer activity extracted from green tea showed a strong affinity to the membrane, which suggests that this significantly limits bioavailability. Ethanol but not dimethyl sulfoxide (DMSO) was found to enhance the diffusion of paracetamol, theophylline, acyclovir, nadolol and amphotericin B. The potential synergistic effect on the diffusion of paracetamol in the presence of caffeine was investigated but it was shown to have a detrimental effect. Finally, an effective protection of Epigallocatechin-3-gallate (EGCG) from the environment was achieved by the preparation of beeswax microspheres as a carrier.

The bioaminergic receptors belong to the G protein-coupled receptors and represent the important drug targets for cardiovascular and neurodegenerative disorders. In this work, molecular dynamics simulations of the bioaminergic receptors in the free form and ligand bound forms have been conducted in the realistic environment in order to address the pharmacological issues such as ligand sub-type selectivity, functional selectivity and selective polypharmacology. Our results provide novel computational protocols that can be used for structure-based drug design of the bioaminergic receptors and are applicable for the structural studies of the other drug targets .

Nitric oxide (NO) has become increasingly recognized throughout the past decade for its role in many patho-physiologies. NO delivery has been investigated as a potential therapeutic strategy against solid tumours, using both NO donor drugs and gene therapy strategies. This project aims to study the importance of oxygen concentration and the possible role of key reaction products involved in NO induced cytotoxicity. Utilising the NO donor drug, DETA/NO the effects of oxygen tension on the cytotoxicity was investigated in vitro. The find ings presented in this study indicate that the DETA/NO mediated cytotoxic effects in all the tumour cell lines tested was significantly more toxic under severe hypoxia. This effect was most evident at drug concentrations greater than 10 ~M. Both the intrinsic and extrinsic apoptotic pathways were activated fo llowing DETA/NO treatment, and cleavage of apoptotic proteins was enhanced under hypoxia. DETA/NO treatment under hypoxia significantly destabilized hypoxia inducible factor (HIF-1a) and resulted in the accumulation of p53 protein. Investigations into the contribution of reactive nitrogen species to NO' cytotoxicity revealed that neither peroxynitrite (ONOO-) nor Nitroxyl (HNO) was generated. Fluorometric analysis in the presence of dinitrogen trioxide (N,O,) scavengers suggests for the first time that N,O, may be responsible for the cytotoxicity with DETA/NO. Compelling evidence is provided to suggest that S-nitrosylation is the key molecular mechanism involved in NO signalling. This is the fi rst study that shows significant Snitrosothiol formation in cancer cells when exposed to high levels of NO irrespective of oxygen concentration. Further investigations revealed that both nuclear p53, and GAPDH proteins are targets for S-nitrosylation following treatment with DETA/NO, and may possibly play a key role in DETA/NO-mediated apoptosis and cytotoxicity.

About 30% of commercial and developmental drugs exhibit poor solubility and thus poor bioavailability. Strategies that enhance solubility of such compounds have become more popular. Cocrystallisation is one of these strategies, so characterisation of in vitro performance of cocrystals is essential. Conventional dissolution systems (USP apparatus 1 and USP apparatus 2) are often not suitable for testing poorly soluble drugs due to failure in providing sink conditions and inability to change the media during the experiment. This project involves designing appropriate dissolution methods that will help understand the mechanism of dissolution of cocrystals. Successful dissolution methods were used to test marketed formulations of Carbamazepine (CBZ) (IR and PR Tegretol® tablets) and Indomethacin (IND) (IR Indocid® capsules). Flow-through cell (USP 4) apparatus proved to be more suitable to test poorly soluble formulations than basket (USP 1) apparatus. Formulations and their cocrystals were tested in four combinations of media: compendial (SGF/SIF), modified I (MGM/MIM-I), modified (MGM/MIM-II) and biorelevant media (FaSSGF/FaSSIF-V2). USP apparatus 4 allowed capturing and quantifying the precipitation of IND samples upon the media change. All Indomethacin formulations exhibited precipitation; however, this occurred into the smaller extent in biorelevant media. The greatest enhancement in CBZ dissolution was observed for Saccharin cocrystals of CBZ. On the other hand, Nicotinamide cocrystal of IND improved dissolution of IND greater than Saccharin cocrystal. Dissolution profiles with physicochemical and pharmacokinetic parameters were used to develop a Physiologically Based Pharmacokinetic (PBPK) model using in silico program Simcyp®. Successful models were then used to predict in vivo performance of cocrystals. Successful PBPK models were developed for IR and PR formulations of Tegretol® tablets and IR Indocid® capsules using dissolution data tested in biorelevant media using dissolution USP apparatus 4. It was found that in vivo absorption of CBZ from cocrystals did not increase dramatically. An overall 2 % increment was observed when compared against the CBZsp sample. However, the rate of absorption for CBZ-SACss samples was significantly faster than the rate of CBZsp absorption. API and cocrystals of IND completely absorbed within 3 hours and IND-NICss cocrystal were found to absorb slightly faster than the other cocrystals. Absorption was 8% higher at 1 h in comparison to IND. However, overall the improvement was not statistically significant. In summary, this research demonstrates that selection of appropriate medium and apparatus is essential to build a successful PBPK model.

Pharmaceutical co-crystals are multicomponent crystalline materials with defined stoichiometry, often stabilized by hydrogen bonding. Numerous studies into co-crystals have been carried out over the last decade or so with the primary aim being to improve the bioavailability of poorly soluble drugs by this crystal engineering technique. However, knowledge gaps remain in understanding the mechanisms by which co-crystals are formed in the solid-state, screening strategies and scale up processes leading to formulation development that are key to success using this approach. The research presented herein mainly focuses upon mechanisms by which pharmaceutical co-crystals are produced in the solid-state. In addition, to protect intellectual property of a drug molecule, co-crystal diversity in terms of polymorphic forms, hydrates/solvates, salt/co-crystals formation, polymorphic co-crystals/salts formation are considered. Hence, an approach towards producing polymorphic co-crystals using different polymorphic forms of an API was studied. Neat grinding and milling were used extensively throughout this work and a novel co-crystal was made which was also found to be polymorphic and distinct from forms manufactured via the liquid state. Methodology solving crystal structures from powder X-ray diffraction data was successfully employed for two co-crystal models. An H-bond propensity calculation was carried out and it is hypothesized that this informatics-based evidence will aid selection of likely co-formers for co-crystals along with an alternative favourable H-bonding scheme for a novel polymorphic co-crystals. Finally, the role of amorphous states in the formation of co-crystals by grinding was found to be a key factor along with energetically and highly kinetically driven situations. The co-crystals formation in the solid-state is a "thermodynamically driven kinetic process" with an apparent "activation energy" needed to be overcome to initiate the process.

Traditionally pharmaceutical manufacture is conducted on a batch basis but significant resources are being invested into the use of intensified continuous processes. This dissertation evaluates the use of a combined twin screw and segmented fluid bed drying process to produce granules on a continuous basis. The experimental program was conducted using structured Design of Experiments in three stages. • Wet granulation only: Investigated the initial relationships between liquid/solid ratio and power required for wet granulation, as well as granule structure using SEM Imaging. • Wet granulation and fluid bed drying: Concluded that the biggest control over, the measured mean granule size (d50) produced from the combined system was still the ratio of water to dry powder in the wet granulation. • Wet granulation through to compression: The effects of changes in the granulation process were not statically relevant on the final tablet for the process set up. The study also used PEPT data to assess motion within the TSG. The studies showed: • The time spent in the kneading zone directly after the liquid addition in relation to the overall time spent in the granulation process appears independent of the process j . set up at 32% ± 2%. • As the barrel speed of the granulator increases the relative time spent in the final ' breakage zone' of the TSG increases, therefore increasing breakage. Using the findings from the literature, the results of the experimental program were used to define the mechanisms occurring within the TSG. The experimental findings were input into a model to predict the outcome of collisions between particles. The model predicts agglomeration of the smaller particles to the larger ones and by calculating changes in the viscosity of the binder the subsequent secondary agglomeration of these granules can also be shown using this model The model is limited due to assumptions in deriving it. The model excludes capillary forces that if given sufficient time to form could have the same order of magnitude strength as other forces.

Interparticulate interactions play a significant role in determining the downstream behaviours of all pharmaceutical formulations and are therefore essential considerations when approaching formulation design. Inhalation product formulation in particular is inherently bound to an understanding of these forces. Delivery of drugs to the lower airways to treat conditions like asthma and COPD requires a particle size of below 5 micron. This implicitly demands micronization of the active pharmaceutical ingredients (APls) and this process renders many particles of large surface area with high surface energies and an auto-adhesive tendency. There is therefore a concurrent reduction in the flowability and dispersion properties of these systems. The interactive character predisposes agglomeration, flocculation or device retention and will compromise manufacture, stability, device function, and the aerosolization behavior of a formulation. Ultimately the ability of any aerosolized API to reach the deep airways is dependent upon adhesion force dynamics. As such, an appreciation of the forces of attraction and scale of particulate interactions within inhaler technology is critical if a successful drug delivery device is to be realized. The advancement of the atomic force microscope (AFM) as a force probing apparatus, has meant that it is now possible to measure the force of adhesion between two particles of interest. However these measurements could not easily be compared, because there is no simple means to account for differences in the contact regime (geometrics) between measurements. However, the development of the cohesive adhesive balance (CAB) approach by Begat, Morton, Stainforth and Price in 2004 has offered a means to negate this limitation. Using a colloidal probe microscopy (CPM) derived technique a particle of a selected material of interest (API, carrier molecule etc.) is attached to an AFM cantilever and ramped onto and off the surface of another material of interest (adhesion measurement), and to a surface of the same material as the tip (cohesion measurement). By graphically plotting the adhesive force values of a series of tips, as a function of the cohesive force values of the same tips, a representation of the relative particle interaction can be obtained. Quantitative information regarding the adhesive/cohesive nature of the interaction can then be extracted from the graph and a description of the interaction formulated that can be compared to other material combinations. The CAB work carried out to date has used recrystallized model substrates. These molecularly flat surfaces ensured there would be no difference between the contact geometry of a functionalised AFM probe and the adhesive and cohesive surfaces of the study respectively. In this fashion the only variable between the two measurements would be the chemical interactivity, and not the interactive surface area. However while using such methodology guarantees the validity of the approach, it is not necessarily a true representation of the materials 'in-situ' and requires more complex sample preparation and complex experimental design. For a variety of reasons this can be misleading in its own right. This thesis details the .investigation into the application of an adapted CAB approach in characterizing the force balance between APls for inhalation in their real state. In so doing, the aim was to see whether such a CAB would offer a quicker and simpler, yet relevant and informative assessment of a drug system force balance. It was hoped that said force balance could in turn be associated with a measurable impact upon the formulation performance of the characterised ingredients as measured 'in-vitro'. This interest was particularly directed at the lesser characterized pressurized metered dose inhaler (pMOI) systems. While these formulations are solvent based, it was of interest to identify whether a simple API to API challenge could infer a descriptive balance that could link to 'in-vitro' performance. Furthermore there was interest in evaluating the use of a range of surface specific imaging techniques to analyse the deposition dynamics of the combination formulations. It was hoped that by doing so, the localisation of the individual components within the binary deposits could again be associated back to the force balance of that system, and that an appreciation of the capability of the techniques involved would be gained. The work that follows therefore commences with the evaluation and description of the capacity for the CAB approach to be adapted to measure force relationships between real beclomethasone dipropionate (BOP) particles and pMDI component surfaces. From this assessment it was found that even with relatively smooth substrates, the combination of bulky functional particles and the inherent substrate roughness caused a critical failure in the CAB model. The parity between cohesive and adhesive geometries of contact was excessively stretched, leading to a loss of force normalisation which was reflected in uncorrelated CAB plots. As a consequence little could be confidently gleaned from the force data acquired, although there was the suggestion that the use of a fluorinated ethylene proplylene (FEP) coating reduced the adhesive interaction between the APls and the pMDI canister wall. This was then followed by an attempt to find a compromise between the model crystal substrates of a pure CAB process and the real particle morphologies that had caused the CAB model to fail. Using a compression process to form API powder compacts, in conjunction with small and discreet functional particles, a confident CAB was achieved for two combinations of APls selected on the basis of surface energy and physical stability analysis. Salbutamol sulphate was characterised with beclomethasone dipropionate, and salmeterol xinafoate with fluticasone propionate. Both combinations showed CAB plots with a sufficiently strong linear regression analysis to suggest a broad accuracy of force balance assessment. Both beta2-agonists showed cohesively dominated relationships with respect to the paired glucocortiocoids, while in reverse both glucocorticoids showed adhesively dominated relationships with the beta2-agonists. There was concern raised over the compression process of the powder discs, and its impact on the physicochemical state of the APls, with some thermodynamic evidence of polymorphic changes that required further work. The next chapter looks at the 'in-vitro' deposition performance of the two API combinations from a HFA134a pMDI system by analysis in an Andersen Cascade Impactor (ACI). The coformulation of salmeterol with fluticasone induced an improvement in the fine particle performance of fluticasone, with a concurrent decrease in the fine particle performance of salmeterol. This impact was hypothesised to be related to alterations in the structure and strength of particle-particle agglomerates. The impact on deposition performance of coformulating beclomethasone and salbutamol was unclear, as a critical unexplained loss of beclomethasone by total recovered mass was seen from all beclomethasone containing formulations. This instability of beclomethasone within the HFA134a system, precluded an accurate assessment of a direct impact on salbutamol deposition. The final chapter, compared a range of surface specific imaging techniques, including scanning electron microscopy (SEM), desorption electrospray ionization mass spectrometry (DESI), Raman spectrometry and time-of-flight secondary ion mass spectrometry (ToF-SIMS) in assessing the extent and nature of 'in-vitro' co-deposition from the salmeterol and fluticasone pMDI formulations. It was apparent that the deposition of the two APls on ACI plates was not likely to be directly comparable assessment of the incidence of particle co-deposition 'in-vivo' due to the forced nature of nozzle directed impaction. However the combination of techniques employed produced a wealth of physical and chemical data that did suggest that the two APls showed extensive co-ordination 'in-vitro'. Raman spectroscopy was able to identify individual particle character and showed frequent salmeterol and fluticasone particle combinations, but suffered from exceptionally long run times and anomalies from photoreactive surface elements. The use of a multivariate approach to ToF-SIMs analysis defined the strong co-association of the two APls, although could not differentiate particle to particle deposition. Multivariate curve resolution (MeR) was used and produced distinct components that segregated ions from both APIS from the background plate but not from each other. SEM imaging was able to define the morphologies of the deposited particles, but was unable to differentiate the two. DES I imaging showed the presence of the two APls together within several drug spots, but could not be used to investigate individual drug spots, and the distribution within, due to inadequate spatial resolution and differences in desorption efficacy. While the co-association of the two APls was observed, the lack of a comparator in another combination of APls made the link between deposition performance and force balance purely descriptive. It was unclear as to whether the force balance of the system lends itself to a particular increase in co-deposition behaviour. However it was apparent that the analytical techniques employed all had respective strengths and weaknesses as mapping tools, and with further work with other formulations could be used to provide a tailored formulation screening process, if subsequent links to force balances could be made.

The weak rheological properties of CS solutions limit their applications in controlled delivery systems. Therefore, polyelectrolyte complex gels were prepared by blending CS with GZ in polybydric alcohols/water vehicles. In addition, CS was blended with PVA in aqueous systems containing dimethylsu1foxide (DMSO). Increasing polymer concentration enhanced the gel properties of the polymer blends. Inclusion of propylene glycol or glycerol in CS/GZ systems resulted in increasing the gel stability. Although glycerol systems showed greater stability than propylene glycol ones, the later exhibited stronger gel properties. The viscoelastic properties of these systems significantly decreased with increasing the pH of the gels resulted in increasing drug release rates. Inclusion of DMSO within PVA/CS systems resulted in their gelation. which increased with increasing DMSO content. The mechanical strength of CS films increased by including PVA, whereas incorporating DMSO resulted in plasticizing effects on the films. However, annealing of the films resulted in increasing their mechanical strength. Inclusion of PVA increased the swelling and hence drug release properties from the films, whereas increasing DMSO content decreased such properties. Another approach used to enhance the mechanical properties of CS films was by preparing interpenetrating networks (IPNs) with poly (hydroxyethylmethacrylate) p(HEMA). The resultant CS/p(HEMA) semi-IPNs exihibited greater mechanical strength comparing to CS films. Coating poiy(methacrylic acid (MAA)-co-HEMA) hydrogeIs by CS resulted in decreasing their swelling properties and hence increasing their mechanical strength. These results may enhance the potential applications ofp(MAA-co-HEMA.) hydrogels in controlled delivery systems. Hydrogels based on p(HEMA) microemulsion networks were prepared using different oils to produce controlled release systems. CS was included within these hydrogels to modify the viscosity of the oil phase and hence to study its effects on the drug release properties. Unexpectedly, inclusion of CS resulted in increasing drug release rates which increased with increasing CS content within the hydrogels.

The accurate prediction of protein-drug binding affinities is a major aim of computational drug optimisation and development. A quantitative measure of binding affinity is provided by the free energy of binding, and such calculations typically require extensive configurational sampling of entities such as proteins with thousands of atoms. Current binding free energy methods use force fields to perform the configurational sampling and to compute interaction energies. Due to the empirical nature of force fields and the neglect of electrons, electron polarisation and charge transfer are not accounted for explicitly. This can limit the accuracy with which interactions are calculated and consequently the free energies obtained. Ideally ab initio quantum chemistry approaches should be used as these explicitly include the electrons. However, conventional ab initio approaches are not suitable due to their prohibitively high computational cost and unfavourable scaling. In this thesis we use large-scale ab initio quantum chemistry calculations within the Density Functional Theory (DFT) method to address the above mentioned limitations of force fields. To obtain quantitative results with ab initio approaches it is important to converge the calculations with the size of the basis set. For this reason we have used the ONETEP program, which is capable of linear-scaling DFT with near-complete basis set accuracy. A well known binding free energy approach is the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA), which obtains free energies from evaluation of the energy of configurations in an implicit solvent model. We present the first application of a “QM-PBSA” approach to a protein-ligand system containing over 2600 atoms. In this QM-PBSA approach the energies of the configurations in vacuum are evaluated with ONETEP. The solvation energies were also obtained with ONETEP using a minimal parameter implicit solvent model within the self-consistent calculation. Large-scale DFT calculations were also applied within a more theoretically rigorous free energy approach which can, in principle, obtain the full entropic contributions to free energy change. The method performs a mutation from a molecular mechanical (MM) description to an quantum mechanical (QM) description of a system. As a result a QM correction is added to the relative binding free energy obtained from a thermodynamic integration calculation within the MM description. This approach was combined with an electrostatic embedding model within ONETEP and used to calculate the hydration energies of small molecules. As well as the computation of more accurate energies, large-scale DFT calculation compute the electron density of the entire system. Using electron density analysis approaches, such as the Hirshfeld density analysis, in combination with energy decomposition approaches, such as a second order perturbation estimate of natural bond orbital interactions, both qualitative and quantitative understandings can be gained into the contributions of particular chemical functional groups that define protein-ligand interactions. These two approaches where applied to study complexes of the Phosphodiesterase type 5 protein and used to rank ligand binding affinities that agree well with then experimentally observed trends.

Within this thesis novel receptors capable of binding and facilitating the transmembrane transport of biologically relevant anions are reported. Molecules based on the diindolylurea and tris(2-aminoethyl)amine scaffolds are described and their anion binding properties in both solution and in the solid state are reported. Inspired by the high affinities for oxo-anions measured for these molecules in polar solvent mixtures, the anion transport properties of structurally simple ureas and thioureas were studied. The high Cl-/NO3 - and Cl-/HCO3 - antiport activity observed with some of the thiourea compounds led to the development of more ‘drug-like’ transporters containing trifluoromethyl substituents. Fluorination of the transporter scaffold in this manner enhanced the lipophilicity and increased the acidity of the NH hydrogen-bond donor groups, leading to improvements in both anion affinity and transport activity. This work ultimately produced compounds capable of facilitating ion transport in vitro. A series of bisurea compounds based on the ortho-phenylenediamine scaffold are reported as potent ion transporters, capable of facilitating a range of ion transport processes. The introduction of electron withdrawing substituents was found to increase transporter activity. Of particular note is a para-nitrophenyl functionalised bisurea that facilitates chloride transport at a loading of 0.1 mmol % (with respect to lipid), the lowest loading of a synthetic mobile carrier to facilitate anion transport reported to date. Dual host systems for both M+/Cl- symport (M = Na, K or Rb) and Cl-/HCO3 - antiport are also described. By using different transporters to facilitate each uniport pathway in these coupled transport processes, it was possible to achieve enhanced ion transport rates. Remarkably, this is the first reported example of a dual host approach towards anion antiport.

The presence of organic salts as drug counter-ions and buffers in hydroxypropylmethylcellulose (HPMC) matrices is often overlooked. This study investigates their potential to influence polymer solution properties and matrix drug release kinetics. A homologous series of aliphatic organic salts influenced solution and matrix properties in rank order of hydrocarbon chain length. Monovalent salts containing 1to4 C-atoms had little effect on polymer surface activity, but lowered sol:gel transition temperatures (SGTT), and accelerated matrix drug release in comparison with a dextrose control. Divalent salts were more potent. These observations are consistent with Hofmeister effects in which anions restructure water in the polymer hydration sheath, induce 'salting-out' and suppressing particle swelling and matrix gel layer formation. Organic salts with StoB C-atoms increasingly influenced polymer surface activity, elevated SGTT, and retarded matrix drug release. This suggests these salts enhance HPMC hydration, possibly through interaction with hydrophobic regions. The effects of these salts on matrix drug release show that these ions impact on water:polymer interactions important to gel layer formation and diffusion barrier properties. HPMC matrices containing SOS and its homologues were also investigated. Turbidimetric, tensiometric and rheological studies supported a mechanism in which these surfactants solubilise HPMC at post-micellar concentrations. Incorporating 10% SOS into HPMC matrices was shown to increase the resistance of HPMC matrices to sucrose medium up to 2.0M, suggesting a role for surfactants in avoiding food solute effects. This study shows that organic salts incorporated in HPMC matrices have the potential to influence drug release in a rank order that reflects their modulation of the HPMC polymer hydration sheath in solution. SOS and its homologous series could retard drug release from HPMC matrices only when their critical aggregation concentration (CAC) was reached. However, it suggests this excipient may have uses as an excipient for improving HPMC matrix release performance.

Currently protein-based drugs are marketed almost exclusively for parenteral administration. Often this may require daily injections which are both painful and inconvenient to the patient, reducing compliance. The application of sustained release technology to proteins offers an improved route of delivery. This can be achieved by encapsulating the protein in a biodegradable polymer. Such systems will reduce the dosing frequency leading to a more efficacious and cost effective treatment. Encapsulating proteins into biodegradable polymers has been found to be promising for the delivery of proteins. Traditional encapsulation methods such as spray drying and emulsion techniques are unsuitable for proteins as they use heat and/or organic solvents, which can lead to the denaturing of the protein. This Thesis describes the use of a supercritical carbon dioxide (scC02) Particles from Gas Saturated Solutions (PGSS) route for the production of protein loaded microparticles. Initially, the performance of a particle production process based on the PGSS was investigated. It was found that post-expansion parameters heavily influence the particle properties in terms of particle yield, size and in vitro drug release. This allowed for the optimum processing conditions for generating the highest yield, smallest particle size and burst release to be identified. Moving on from this, the following chapter focuses on the particulate formulation by investigating the effect of blending different polymers to improve the particulate properties. Particles were produced from a base formulation of poly(1actic acid) (PLA) and poly(1actic-co-glycolic acid) (PLGA) blended with various excipients such as Poloxamer 407, poly(ethyl glycol) (PEG), poly(propylene glycol) (PPG) and a CO2-philic excipient PVAc-co-PVPi. It was found that increased miscibility of the excipients in the base formulation improves the compatabilisation of the protein and the polymer matrix, leading to a more homogeneous protein distribution in the polymer matrix. A more homogenous protein distribution within the microparticle was found to improve the in vitro release properties. The final chapter of this Thesis using a statistical Design of Experiment (DoE) approach to further investigate the effect of the excipient Poloxamer 407 when blended with PLA and PLGA. The formulation was optimised to produce a very small particle size of 31 f!m enabling successful injection through a target 25 G needle.

Nuclear receptors (NRs) are biomedically important transcription factors that regulate gene expression by recruitment of coactivators and corepressors (cofactors) to target gene promoters. Humans express 48 different NRs.and their isoforms, approximately half of which are orphans that have no recognised ligand. NRs can interact with more than 350 known cofactor proteins, many of which are chromatin modifying enzymes. Binding of ligand induces a conformational change in the NR that stimulates or prevents the docking of cofactors. These interactions are mediated by signature motifs (LXXLL in coactivators; or LXXXIXXXI/L in corepressors) that are essential for NR/cofactor function. To allow broader understanding of cofactor selectivity, an NR LBD interaction panel was constructed consisting of seven ligand-binding and eighteen orphan NRs. Interaction studies using LXXLL motifs from the well-characterised cofactor SRCl and the lesser-studied cofactor MEDl identified distinct patterns of interaction within Class I and Class 11 subsets of NRs. Novel motifs within the developmental regulator BCLllA, with consensus Y /FSXXLXXL/Y, were also investigated, revealing selective binding to a group of related orphan NRs consisting of the NR2E/F subfamilies. This sequence was also found to be conserved in other NR cofactors such as NSDl and was again shown to facilitate interactions with this subset of orphan NRs. As highly social transcription factors, nuclear receptors form a complex and integrated dimerisation network, binding to DNA as monomers, homodimers and heterodimers. While heterodin:terisation of nuclear receptors remains poorly understood, it is known to increase the complexity of NR-mediated transcription by integrating gene networks, mUltiple ligand inputs, cofactor selectivity, and increasing competition for other heterodimeric partners. Having determined the cofactor binding preference of the NR2E/F subfamily we profiled their dimerisation, revealing diverse dimerisation properties and several interactions of interest, including novel complexes of PNR that are likely to be of physiological consequence in the retina. ii

Recent studies have shown the numerous advantages associated with specific drug delivery to the colon, highlighting its favourable conditions and long transit time as the main advantages. A number of in vitro studies also show that the delivery of liposomes to the colon could provide further advantages due to bonding to the colonic mucosa in both healthy and inflamed regions. Despite these apparent advantages no oral liposomal formulation has been developed for targeted delivery to the colon as yet. Initially, experiments were conducted in which liposomes were directly coated with the pH responsive polymer Eudragit S100. Although the coating was shown to slow drug release in simple pH buffers, it was realised it could not protect the lipid membrane from the model bile salt sodium taurocholate. Development of the formulation moved onto the production of Eudragit S100 microspheres to provide a solid barrier to protect the liposomes. Due to the solvents required in the microsphere production it was essential to protect the liposomes, which was done by coating them with the enzyme controlled polymer chitosan. The final stage involved encapsulating chitosan-coated liposomes within the Eudragit microspheres to produce a novel, colon targeting liposome-in-microsphere (LIM) formulation.

Dry powder inhaler (DPI) formulations are usually comprised of a mixture of micronised active pharmaceutical ingredient (API) with aerodynamic diameter 1-5 m to allow deposition in the lower airways, and a coarse (~70 micron) excipient, typically \(\alpha\)-lactose monohydrate, used to aid the handling, metering and dosing of the formulation. These components are usually combined in a secondary manufacturing process such as high shear blending (HSB), which is used primarily to distribute the cohesive drug particles throughout the bulk excipient to create a chemically homogeneous formulation. This thesis explores the use of HSB to produce mimic DPI formulations and assesses the effect of different blending and storage regimes on various physicochemical properties of such powders. A novel fluidised bed elutriation (FBE) technique was developed to test the ability of fine mimic drug particles to separate from coarse particles in such formulations, along with conventional air jet sieve (AJS) and Next Generation Impactor (NGI) studies. Results showed that, generally, blending regimes were seen to have little effect on the in vitro performance of these mimic formulations, with extended storage at high humidity having a more profound effect on the separation of fine particles. Tests on the performance of formulations using the FBE technique showed that fluidisation performance alone is insufficient to identify blending-induced changes; however, analysis of the fine elutriated fraction has shown subtle changes in the populations of particles due to HSB. Notable differences were observed between the mimic cohesive and adhesive drugs, indicating the ability of these tests to identify formulations with different adhesive properties. In addition, a study to determine the specific energy input (SEI) required to achieve chemical homogeneity suggested that extended high shear blending beyond a given energy input may only alter the size distribution of the formulation, and not improve homogeneity, thus having implications for the manufacture of such products.

Glycosides play a major part in the development of novel pharmaceuticals since the sugar ligands of natural products play important roles in, e.g., cell-wall synthesis. The use of phosphosugars in the pharmaceutical industry has been linked to anti-inflammatory and immunosuppressive drugs. Therefore it's of interest to be able to develop and manufacture unique glycosylated natural products and investigate their abilities as potential drugs. A technique called In Vitro Glycorandomization (IVG) has been developed for the production of novel varieties of the glycopeptide antibiotic vancomycin. IVG need a range of sugar-l-phosphates for the production of different NDP-sugars. An enzyme that can phosphorylate a wide range of sugar substrates at position 1 is of importance to simplify the technique. Two human enzymes, galactokinase and N-acetylgalactosamine kinase, were mutated at specific residues in their amino acid sequence in an attempt to increase the number of sugars each enzyme could phosphorylate. Galactokinase showed activity toward two sugars not including its natural substrate galactose: 2-deoxy-D-galactose and D-galactosamine-hydrochloride. The promiscuity of galactokinase was increased after mutating the tyrosine at position 379. The Y379W mutant was the most successful by phosphorylating seven of the nine sugars selected for this enzyme. Several N-acetylgalactosamine kinase mutants showed higher activity toward the natural substrate (N-acetylgalactosamine) but, overall, the wild type was the better enzyme for the three selected sugars for this enzyme. Since no structural changes were visible when performing homology modelling of the mutants it is suggested that the increase in promiscuity for galactokinase is due to a rearrangement of hydrogen bonds within the proteins. It was shown that the flexibility of the galactokinase enzymes declines when substrates are bound in the active site and the mutants became more rigid than the wild type. The mutants are, however, more flexible than the wild type before the substrates bind.

Targeting the minor groove of DNA with small molecules is an important recognition strategy in biology. A wide range of minor groove binding ligands (MGBLs) with good sequence discrimination ability are of interest as potential therapeutic agents in a variety of human diseases such as cancer, along with anti-bacterial and/or anti-parasitic activities. Whilst the detailed mechanism of action of some of these MGBLs is still unproven, they are known to be effective inhibitors of a number of minor and major groove binding protein-DNA interactions. This thesis reports on crystallographic studies to determine the molecular structure of MGBLs bound to DNA sequences, in order to better understand the details of their molecular recognition by DNA. Several interesting MGBLs differing in their structural features were crystallised with A-T rich oligonucleotides for neutron and high-resolution X-ray data collection. Phases for the X-ray crystal structures were determined using molecular replacement, with diffraction data up to 1.2 A resolution. The crystal structure revealed the MGBLs bound in the central AATT or AAATTT rich region of the minor groove of the DNA. The ligands form hydrogen bonds with the bases of the DNA at the floor of the minor groove directly or mediated via water molecules depending on the shape of the ligand. Several oligonucleotide-MGBL complexes were crystallised in the presence of deuterium oxide (heavy water) with the aim of studying the water network around the minor groove in the presence of ligand using neutron crystallography. In order to further our understanding of the biological mechanism of action of MGBLs, biophysical studies were undertaken with the DNA major groove binding transcription factor, NF-κB. This transcription factor binds to the continuous guanine and cytosine bases of the major groove leaving the minor groove exposed to other molecules. Surface plasmon resonance (SPR) and small angle X-ray scattering (SAXS) studies were undertaken to study the effects on MGBLs on NF-κB-DNA binding. It was revealed that MGBLs had significant effect on the protein-DNA interactions which was further dependent on the shape of the MGBLs.

This research was initiated as part of the fight against the public health problems of rising counterfeit, substandard, and poor quality medicines and herbal products in the Malaysian market. A simple, quick and cost-effective drug screening procedure using an incremental near infra-red [NIR] spectral database of common medicines in combination with principal component analysis [PCA] was developed to facilitate drug analysis without depending on standard compounds or products from manufacturers. The novelty of the approach is demonstrated by this two-tier method which allowed application in product identification, drug quality study, herbal analysis, and the detection of counterfeit and adulterated medicines. The NIR spectra database consisted of almost 4,000 spectra from 15 types of medicines and 3 types of herbal preparations, acquired and stored in the database throughout the study. The optimization procedure developed in this thesis on the database produced a search strategy using correlation and first derivative correlation algorithms on the full spectrum. The cut-off points of the hit quality index (HQI) were determined to classify the unknown sample in four categories; similar batch/match (classification type 1, < 0.0001), same brand/different batch (classification type 2, < 0.01), same type of medicine/different brand (classification type 3, < 0.1) and different type of medicines (classification type 4, > 0.2). The same set of spectra were analysed by multivariate methods, including PCA. This two-tier screening approach proved successful when challenged firstly using simple compound drugs followed by complex mixtures of herbal preparations and then using alleged counterfeit and adulterated samples seized by the authority. The method developed has allowed samples to be identified without known background information which was difficult using other qualitative NIR techniques that required reference products for comparison. It is also allowed selection of suspected samples for further extensive analyses and in detection of adulterant contaminating herbal preparations. The outcomes of this thesis should support further qualitative and quantitative researches on drugs and herbals products in Malaysian universities. The developed spectral database of drugs will providea valuable tool to assist in drug quality surveillance nation-wide.

The process of pharmaceutical tablet dissolution is a vital stage in the delivery of active pharmaceutical ingredients (APIs). The constituent components and their spatial arrangement within the tablet determine the release characteristics of the API. It is therefore important to understand and characterise the various processes and component interactions that occur during tablet dissolution. Computational simulations of tablet dissolution can be used to obtain parametric sensitivities and optimise formulations so that the desired API release profile is achieved. This thesis describes the methods behind modelling the behaviour of non-swelling and swelling tablets, the mathematical validation of the models, parametric studies and the experiments which were used to obtain parameters and verify the models. The experimental method used in this work is Fourier Transform Infrared (FTIR) spectroscopic imaging, which, when using an attenuated total reflection (ATR) accessory and flow cell, enable chemical and spatial information to be obtained from the tablet as it dissolves. UV/Visible spectroscopy was also used to obtain drug release information. The non-swelling model discretised a tablet over a Cartesian grid and solved the mass transfer equations (dissolution and diffusion) to obtain drug release profiles. Two parametric studies were conducted where the particle size distribution and mass fractions were varied in one, and the API diffusivity, saturated concentration and mass fraction in the other to see what effect these had on drug release, demonstrating the importance of the choice of excipient and the impact of particle size on release variability. For experimental validation, tablets containing different quantities of polyethylene glycol and nicotinamide were dissolved and imaged, and optimisation was used to obtain the pure component saturated concentrations. The model was then tested against a different tablet to demonstrate the predictive capability of the model. The swelling model discretised a tablet into small cylindrical particles, whose size was proportional to the mass of components within them and whose motion was determined using the Discrete Element Method (DEM). As water diffused into polymer particles, they could expand, resulting in macroscopic swelling. The DEM model of a swelling and dissolving tablet was validated against a numerically exact model of the same tablet and parametric studies were conducted into the effect of polymer disentanglement threshold, polymer equilibrium water fraction and polymer dissolution rate. The model was also optimised against a dissolving tablet containing HPMC to obtain parameters for this excipient. To conclude, both models were implemented, validated and found to accurately describe the dissolution kinetics of both swelling and non-swelling tablets.

The pharmaceutical industry is currently facing an industry wide problem of high attrition rates for new compounds and rising development costs. As a result of this, there is an emphasis on making the development process more ecient. By learning more about new compounds in the early stages of development, the aim is to stop ineective compounds earlier and improve dose selection for compounds that progress to phase III. One approach to this is to use adaptive designs. The focus of this thesis is on response adaptive designs within phase IIb dose-finding studies. We explore adapting the subject allocations based on accrued data, with the intention of focusing the allocation on the interesting parts of the curve and/or the best dose for phase III. In this thesis we have used simulation studies to assess the operational characteristics of a number of response adaptive designs. We found that there were consistent gains to be made by adapting when we were relatively cautious in our method of adaptation. That is, the adaptive method has the opportunity to alter the subject allocation when there is a clear signal in the data, but maintains roughly equal allocation when there is a lot of variability in the data. When we used adaptive designs that were geared to randomising subjects to a few doses, the results were more varied. In some cases the adaptation led to gains in efficacy whilst in others it was detrimental. One of the key aims of a phase IIb dose-finding study is to identify a dose to take forward into phase III. In the final chapter, we show that the way in which we choose the dose for phase III affects the expected gain, and so begin to consider how we can optimise the decision making process.

Modern day medicine is on the brink of a new age of therapy, which aims to harness the natural power of molecular biology for disease treatment. This therapy could include replacement of dysfunctional genes that cause disorders such as cystic fibrosis (Lommatzsch and Aris, 2009), or silencing the overexpression of genes that cause disorders such as cancer (Pelengaris and Khan, 2003). In both examples, the treatment of these genetic diseases lies in the delivery of synthetic nucleic acids into diseased cells, the former being called gene replacement therapy (Dobson, 2006a), and the latter being called RNA interference (RNAi) therapy (Whitehead et al., 2009). While these techniques have long been in use as genetic research tools for gene transfection or silencing in vitro, their translation for use in clinical disease treatment has yet to be achieved. The main problem facing the development of these novel therapies is the specific delivery of nucleic acids into diseased cells within the body. It is hoped that nanoparticles (NPs) can be used to overcome this problem, by acting as vehicles to transport nucleic acids through the body for specific delivery into diseased cells. This feat can be aided by the attachment of additional functional molecules such as cell penetrating peptides (CPPs), targeting peptides, additional drug types and molecules for imaging during treatment. Many different NP design strategies are currently under development. It is essential for new designs to be extensively tested for toxicity and efficiency in human cells before they can be successfully released into the clinic. As part of this effort, this PhD project has investigated two different NP design strategies for drug delivery: 1) the use of a magnetic field (MF) and a CPP to increase the delivery of iron oxide magnetic NPs (mNPs) to cells grown in tissueequivalent 3D collagen gels, and 2) gold NPs (AuNPs) for the delivery of siRNA to silence the c-myc oncogene for cancer treatment. In the first investigation, a MF and the CPP penetratin were found to increase mNP delivery to cells grown in 3D. In the second investigation, AuNPs were assessed in a range of different cell types (grown in 2D) for their performance in 4 main areas; cellular toxicity, cellular uptake, c-myc knockdown and effect on the cell cycle.

The primary purpose of this study was to investigate the combination of experimental and computational methods in the search for reproducible colloidal surface-enhanced Raman scattering of pharmaceutical compounds. In the search for optimal experimental conditions for colloidal surface-enhance Raman scattering, the amphipathic β-blocker propranolol was used as the target molecule. Fractional factorial designs of experiments were performed and a multiobjective evolutionary algorithm was used to find acceptable solutions, from the results, that were Pareto ranked. The multiobjective evolutionary algorithm suggested solutions outside of the fractional factorial design and the experiments were then performed in the laboratory. The results observed from the suggested solutions agreed with the solutions that were found on the Pareto front. One of the experimental conditions observed on the Pareto front was then used to determine the practical limit of detection of propranolol. The experimental conditions that were chosen for the limit of detection took into account reproducibility and enhancement, the two most important parameters for analytical detection using surface-enhanced Raman scattering. The principal conclusion to this study was that the combination of computational and experimental methods can reduce the need for experiments by > 96% and then selecting solutions from the Pareto front improved limit of detection by a factor of 24.5 when it was compared to the previously reported limit of detection for propranolol. Using the same experimental conditions that were used for the limit of detection, these experiments were extended to plasma spiked with propranolol in order to test detection of this pharmaceutical in biofluids. Concentrations of propranolol were prepared using plasma as the solvent and measured for detection using colloidal surface-enhanced Raman scattering. Detection was determined as <130 ng/mL, within physiological concentrations, previously achieved using separation techniques. The second part of this thesis also involved a combination of experimental and computational methods. Raman optical activity was utilized to investigate secondary structure of amino acids and diamino acid peptides in combination with density functional theory calculations. Amino acids are important biological molecules that have vital functions in the biological system. They have been recognized as neurotransmitters and implicated in neurodegenerative diseases. Raman and Raman optical activity experimental results were compared to determine site-specific acetylation, marker bands for constitutional isomers and identification of functional groups that interact with the solvent. The experimental spectra were then compared to those from the density functional theory calculations. The results indicated that; constitutional isomers cannot be distinguished from the Raman spectra but can be distinguished from the Raman optical activity spectra, site-specific acetylation can be identified from the Raman spectra, however, Raman optical activity provides more structural information in relation to acetylation. When the results were compared to the density functional theory calculations for the diamino acid peptides the results agreed reasonably well, however, agreement was not as good for the monoamino acids because diamino acid peptides support fewer conformations due to the peptide bond whereas monoamino acids can adopt a far greater number of conformations. Combined computational and experimental techniques have developed the ability to detect and characterize biomedical compounds, a significant move in the advancement of Raman spectroscopies.

Today, drugs used in treating disease are almost all hydrophobic, which makes them unattractive for applying to patients. To overcome this problem, an amphiphilic macromolecule was designed which can deliver drugs to the specific targeted area. Therefore, this study focuses on the synthesis of water soluble hyperbranched polymers and their application in drug delivery systems. The first part of the thesis describes the synthesis of water soluble hyperbranched polymers by the anionic-polymerisation technique, with p-nitrophenol as the core and glycidol as the monomer. The core is important to control the molecular weight of the hyperbranched polymer. We then produced five different molecular weights by varying the core to monomer ratio, which produced hyperbranched polymers with molecular weights of 4000 Da, 8000 Da, 12500 Da, 27000 Da and 50000 Da, and four different concentrations of each molecular weight, 1.00 x 10-4 M, 2.00 x 10-4 M, 4.00 x 10-4 M and 6.00 x 10-4 M. To investigate the performance of the hyperbranched polymers, four different hydrophobic molecules were studied. The molecules were naphthalene, ibuprofen, tetracarboxyphenyl porphyrin (TCPP) and anti-prion drug. Encapsulation studies showed the concentration of each molecule increased with an increase in molecular weight and concentration of the hyperbranched polymer The functionalisation of folic acid on the surface of the hyperbranched polymer would enhance the effectiveness of the polymer as a drug delivery agent for targeting in cancer treatment. The above macromolecule was synthesised by conjugation with folic acid through poly (ethylene glycol) as spacer. The final chapter of this work describes the encapsulation of two hydrophobic molecules, ibuprofen and tetracarboxyphenyl porphyrin with water soluble PAMAM dendrimers. PAMAM dendrimers from G0.5 to G2.5 were synthesised. The results showed that more drug solubilised, the concentration of both molecules increased with an increase in dendrimer concentration. Then a comparison was made between encapsulation with water soluble hyperbranched polymers and water soluble PAMAM dendrimers. Similar molecular weight was used for both polymers of 4000 Da. For both polymers, the result demonstrated that the concentration of drug rose at 2.00 x 10-4 M and plateaued at a polymer concentration of 6.00 x 10-4 M. It was suggested the above trend was due to the aggregation of polymer. The above studies make a valuable contribution to the development of good drug delivery systems.

Biodegradable microneedles (MNs) are currently being developed to painlessly facilitate the effective permeation of therapeutic substances across the skin barrier. As sugar glasses are utilised in nature to protect proteins and other delicate structures upon dehydration, such materials may be an appropriate substrate for the preparation of biodegradable MNs. The aim of this work was to investigate for the first time the feasibility of preparing biodegradable MNs from sugar glasses and to test their potential utility for drug delivery applications. Solid sugar products were fabricated from 32 different solutions containing a range of individual sugars and binary sugar combinations, utilising a low temperature dehydration methodology. Subsequently, a novel vacuum-forming micromoulding methodology was developed and optimised to produce sugar glass microneedle (SGMN) arrays from silicon master structures. The sugar materials and MN structures were characterised using a variety of microscopic, thermal and x-ray diffraction analyses. The ability of SGMNs to puncture human skin was assessed in an in vitro skin model, whilst SGMN facilitated drug delivery was investigated using modified static Franz-type diffusion cells. A range of model substances including methylene blue (MB) dye, ibuprofen sodium (IBU), sulforhodamine B (SRB), FITC-BSA and β-galactosidase (β-gal) were incorporated within SGMN arrays. Furthermore, novel SGMN adhesive patches containing SRB within the backing only were fabricated using silicone and acrylate adhesives. Long-term stability of SGMN arrays was assessed under a range of differing storage conditions. Initial characterisation studies suggested that non-crystalline sugar material was formed from anhydrous trehalose and sucrose (75:25 %w/w) sugar solutions. This finding was critical to future SGMN fabrication and incorporation of model substances within the material. Process optimisation led to fabrication of SGMNs with strong morphological fidelity to master structures, which reliably penetrated human skin to facilitate diffusion of MB dye. Furthermore, SGMNs were shown to dissolve rapidly and completely in human skin and deliver MB, IBU, SRB and FITC-BSA to the deeper skin layers. Diffusion study data suggested that SGMN arrays incorporating a range of model substances facilitated permeation across skin in a bolus delivery manner. Additionally, it was found that SGMN adhesive patches were able to control permeation of SRB, a model hydrophilic compound. Sugar glasses containing β-gal were shown to stabilise enzyme functionality at approximately 40 % of initial activity over a 3 month period when stored under desiccation. Elevated humidity and temperature storage was detrimental to SGMN morphology, with 10 % relative humidity at 20 °C being optimal for MN preservation. Overall, this study suggests the utility of SGMNs for the stable incorporation and effective intra- or trans-dermal delivery of a range of model substances, including hydrophilic and macromolecular molecules. Furthermore, it was shown that a novel SGMN adhesive patch may provide the capability to control drug release across skin. Sugar glasses demonstrated a stabilising effect upon a functional protein cargo, although it appeared that storage conditions had a strong influence upon physical SGMN stability.

Both excitable and non-excitable cells possess plasma membrane ion channels and evidence has accumulated over the last 30 or so years that these channels perhaps play key roles in the cell life and death. This Thesis investigated the characteristics and putative functions of one class of potassium channel, the BK channel in osteoblast-like cells and primary osteoblasts from human, rat and mouse. The properties and functions were defined in vitro using a combination of patch-clamp, reverse transcription-polymerase chain reaction (RT-PCR) and functional assays for cell growth and mineralisation. RT-PCR showed the presence of KCNMA1, KCNMB1, KCNMB2, KCNMB3 and KCNMB4, the gene for BK channel α, β1, β2, β3 and β4 subunits respectively. The channel was voltage-dependent with a mean unitary conductance of 315 pS in cell-attached patches, a conductance of 124 pS in excised outside-out and 151 pS in inside-out patches. The channel was blocked by TEA (0.3 mM), TBuA (1 mM), TPeA (1-10 μM), THeA (1-3 μM), tetrandrine (5-30 μM) and paxilline (10 μM) and was activated by isopimaric acid (20 μM). Notably iberiotoxin (IbTX) (90 nM) only blocked a proportion of the channels tested (2/5). Osteoblast-like MG63 cell number changed in response to BK channel modulators. It increased significantly with TEA and tetrandrine at low concentrations (1 mM, 3 μM respectively), and reduced at high concentrations (>10 mM, >10 μM respectively). It was not affected by IbTX (20-300 nM) or slotoxin (300 nM). The increase in cell number by TEA was blocked by isopimaric acid. In addition, TPeA and THeA caused a decrease of osteoblast-like SaOS2 cell mineralisation at the concentrations (3 and 0.3 μM, respectively) increased MG63 cell numbers. The BK channel has a distinctive pharmacology and represents a new target for therapeutic strategies in modulating osteoblast proliferation.

The existence of functional selectivity at the mu-opioid receptor was examined by determining the efficacy of a range of opioid agonists for promoting G protein activation and arrestin-3 translocation. In general, there is a good correlation between the efficacy of an opioid agonist at promoting G protein signaling, and the efficacy at recruiting arrestin-3 to the receptor. Endomorphin 2 appears to be an example of a biased ligand with significantly higher efficacy for arrestin-3 translocation, while morphine does not appear to be biased. The kinetics of binding for DAMGO, morphine and endomorphin 2 were determined by competition kinetic assay as a potential explanation for the apparent bias of endomorphin 2. Mean occupancy times of DAMGO, endomorphin 2 and morphine at MOPr are similar to the time required for GRK2-mediated phosphorylation, indicating that kinetics of binding may be a determinant of their ability to promote arrestin-3 signaling at MOPr. The abilities of DAMGO, morphine and endomorphin 2 to induce desensitization of GIRK currents in AtT-20 cells expressing wild type mu-opioid receptor, mu-opioid receptor containing S261/363A substitutions, and mu-opioid receptor with a C terminal truncation from amino acid 354-398 were examined. From the results, it appears that agonist-induced acute desensitization in AtT-20 cells has multiple components. C terminal truncation of MOPr resulted in a slight inhibition of endomorphin 2-induced desensitization. Alanine substitution at serine 261 and 363 inhibited desensitization induced by endomorphin 2. Treatment with the GRK2 inhibitor 5-[2-(5-nitro-2- furyl)vinyl]-2-furoate slightly but significantly inhibited desensitization induced by DAMGO, and to a very small extent morphine, but not endomorph in 2, which may indicate that biased ligands trigger receptor regulation in a different manner to unbiased ligands.

Polymorphism presents complex issues for the pharmaceutical industry from processing, regulatory, patenting and stability perspectives. It can be further challenging to control the same form throughout processing and development when it has the capacity to form a hydrate. Incorporation of water into the crystal lattice contributes to significant differences in solubility, stability and bioavailability of the active pharmaceutical ingredient (API). During processing and formulating steps, water is used in many procedures such as, recrystallisation, wet granulation, aqueous coating lyophilisation etc. This can trigger anhydrous to hydrate conversion and could be detrimental for bioavailability and stability of the product. The factors responsible for this type of transition such as, role of solvent, activity of solvent, thermodynamic stability of different forms, equilibrium conditions, processing induced transformations are investigated. Theophylline, a channel hydrate, is chosen as a model compound which exhibits both polymorphs and solvates. The value of water activity at which the theophylline monohydrate is thermodynamically stable form was investigated using solubility, cooling crystallisation and slurry experiments and found to be aw 2: 0.70 at 25 QC. Full characterisation of the solid state chemistry of theophylline has resulted in the discovery of a new, previously unreported, anhydrous form of theophylline, called Form IV. Using solubility, crystallisation, slurrying and thermal experiments, Form IV was found to be thermodynamically more stable than the currently known stable form, Form H. The crystal structure of Form IV and Form I was determined by single crystal :X-Ray diffraction technique. The crystal structures for Form IV and Form I are deposited in Cambridge Structural Database (CSD) with reference code BAPLOT03 and BAPLOT04 respectively. The experimentally observed stability behaviour was correlated with the structural features of solid forms and also with the energy calculations. The kinetic ally stable Form H serves as the intermediate for polymorphic and hydrate-anhydrate transformations as the catemer motif observed in Form II can easily propagate by forming a strong and directional hydrogen bonds. In contrast, the dimer of theophylline molecules as observed in Form IV needs the presence of solvent to link through other dimers only by weak interactions. This results in the generation of Form IV only via solvent mediated transformations. Solid state chemistry of hydrate forming compounds Theophylline has also been used here as a model compound to study eo crystallisation with various saturated, dicarboxylic acids. A new, eo crystal of theophylline with adipic acid was generated and using thermal methods and PXRD, the stoichiometry (1 :2, adipic acid: theophylline) is confirmed. The complex hydration-dehydration behaviour of theophylline was investigated. The samples subjected to different pharmaceutical processing conditions for hydration-dehydration, generated various .intermediate phases suggesting multiple dehydration mechanisms and the potential of phase transformations during processing of such kind of hydrate forming compounds. The sensitivity of thermal methods over other bulk methods such as PXRD, in detecting a small amount of phase impurity, has been highlighted.

The use of polar anti-solvents for the crystallisation of lactose from 10% (w/v) aqueous solutions has been investigated. Crystal growth was observed at 50-65% antisolvent content and showed a morphological transition from polyhedral to needle-like habit with increasing antisolvent content, which coincided with a polymorphic transition from alpha lactose monohydrate (t:a. H20) to beta lactose (L~). Where anhydrous dehydrating antisolvents were employed such a~ methanol and ethanol, evidence of La. H20 dehydration to form stable anhydrous alpha lactose (Las) was also observed at 95% antisolvent content. Powder X-ray diffraction (PXRD) analysis of the samples highlighted the preferred orientation effects exhibited by large crystals of this kind, indicating the difficulties experienced by the non-specialist when performing phase identification of lactose polymorphs by PXRD. Application of the same crystallisation procedures to a racemic mixture of the active pharmaceutical ingredient (API) salbutamol sulfate (SS) indicated that some conditions can promote the formation of solvated SS. Ethanolic suspensions of spray dried and micronised La. H20, with average particle size between 3 and 200~m, were prepared by static and reflux methods and compared with sub-micron lactose (SML) suspension prepared by a high pressure homogenisation approach. Dehydration behaviour as a function of time was investigated by 13C CP MAS NMR spectroscopy and in all cases, indicated that suspensions contained Las. Several approaches were employed to remove ethanol from these suspensions and the products were analysed by PXRD and scanning electron microscopy (SEM). For samples with mean particle size greater than one micron, Las was retained on removal of the ethanol, although differences in the morphology and particle size of the crystals were apparent. These data imply that while SML is stable under dry conditions it is more susceptible to rehydration than standard Las with particle size between 3 and 200~m. in-situ 13C CP MAS NMR spectroscopy, employing hand-made glass inserts, was used to investigate the dehydration behaviour of La. H20 to Las in ethanolic suspension. Strong ethanol 13C resonances were observed for some samples, indicating a liquid-solid interaction between the ethanol and lactose surface. Replacement of ethanol with anhydrous methanol, n-butanol and 3-methylbutan- 2-01 implied that the solvent mediated dehydration is sterically controlled. 13C CP MAS NMR studies of SS, fluticasone propionate (FP) and salmeterol xinafoate (SX) showed that both SS and FP exhibit very short relaxation times for a solid material (-2s). This liquid-like behaviour is particularly beneficial pharmaceutically, as these APls are often mixed with an excess of lactose in inhaled formulations. Lactose behaves as a typical crystalline solid, and therefore experiences significantly longer relaxation times (-360s). Signals from SS and FP were successfully isolated from lactose/API blends (98%/2%) simply by reducing the delay times used within the 13c CP MAS NMR experiment.

Crystallization is a widely used purification and separation technique in the pharmaceutical industry. More than 90 % of the active pharmaceutical ingredients are produced in the crystalline form. The quality of the crystalline product greatly affects the downstream processing and bioavailability of the drug. The Food and Drug Administration (FDA) initiated in 2004 the use and implementation of process analytical technology (PAT) in the pharmaceutical development and production and encourages the pharmaceutical industry to adopt quality by design (QBD) approaches. The prime objective of this initiative has been to optimize the drug development and manufacturing process by reducing cost, improving product quality and reducing the number of failed batches. The work presented in this thesis focuses on expanding the use of two PAT tools, namely attenuated total reflection ultra violet/visible spectroscopy (ATR-UV/Vis spectroscopy) and focused beam reflectance measurement (FBRM). ATR-UV/Vis spectroscopy and FRBM are mostly used for process monitoring. The aim here was to develop sophisticated control approaches using these in situ tools for enhancing the product quality. Chemometrics is an integral part of PAT, and can provide valuable information about the system. This tool has also been used in this study for calibration model development and monitoring the cooling and antisolvent crystallization processes for single and muticomponent crystallisations. The development of an accurate and robust calibration model is necessary for qualitative and quantitative analysis of a system using spectroscopy. A systematic methodology was therefore presented for the selection of a suitable calibration model for ATR-UV/Vis spectroscopy. The developed model was then used as part of supersaturation control approach (SSC). SSC uses information from ATR-UV/Vis spectroscopy in a feedback control loop to keep the system at desired supersaturation. The developed approach resulted in the production of crystals of uniform size and can represent the bases for a model-free direct design approach for crystallization systems.

Diphenylalanine (FF) peptide is the core-recognition motif of β-amyloid polypeptide, a peptide associated with diseases such as Alzheimer’s and which is known to be capable of self-assembly. FF has attracted interest in nanotechnology due to the physical and chemical stability and mechanical rigidity of the self-assembled nanotube form of the peptide. A number of promising applications of FF nanotubes have previously been explored. To extend this work to biomedical and pharmaceutical areas, an improved understanding of the physicochemical properties of FF tubes, together with the influence of assembly conditions, cytotoxicity properties and potential in drug delivery field are presented in this thesis. The studies presented in Chapter 2 address the self-assembly of FF peptide prepared by two known methods of preparation, one aqueous based, the other utilizing an organic solvent. A range of complementary characterization methods is applied including atomic force microscopy, scanning electron microscopy, focused ion beam-scanning electron microscopy, X-ray powder diffraction, and Raman Spectroscopy. The investigations reveal differences in morphology of the tubes formed by the different preparation methods. The aqueous based method produces tubes that are long, straight and unbranched and are consistent with previous work. The alternative organic solvent method produced tubes that are shorter and narrower. In addition, these tubes displayed flexibility and nucleation points. Following on from these findings, a proposed mechanism of tube growth is discussed. Chapter 3 further extends the investigation to the biological field. Possible cytotoxicity issues are studied using a MTT assay on a HeLa cell line. Moreover, total internal reflection microscopy was applied to investigate HeLa cell behaviour in the presence of FF nanotubes. The results from these studies reveal that the nanotubes and FF peptide do not cause any mitochondrial related damage to HeLa cells. Furthermore, short tubes were observed to be taken up by cells through a suggested macropinocytosis pathway. Finally, in Chapter 4 the focus turns to the investigation of the potential of FF tubes as drug carriers in drug delivery. Here, successful synthesis of drug-loaded FF tubes is presented with two model drugs. The physical characterization of the complex formed under different conditions using scanning electron microscopy reveals FF nanotube self-assembly is a drug concentration and solvent type dependent process. Finally, in vitro drug release from FF nanotubes is performed and compared to that of the drug alone. Extended drug release is observed for both drug candidates and release mechanisms are proposed. The results presented throughout this thesis demonstrate the versatility of self-assembling FF peptides for the formation of tubular nanostructures with different morphologies and physical properties under different conditions. The assembled nanostructures appear non-toxic to cells and offer promise in drug delivery as novel drug carriers.

In recent years the pharmaceutical industry has seen a rise in the number of drug compounds with low aqueous solubility, and consequently poor oral bioavailablility. One potential solution to this problem is to formulate such compounds as solid dispersions, whereby the drug is dispersed in a carrier matrix in the solid state. In this thesis, the hypothesis that a number of drug-drug and drug-polymer intermolecular interactions influence the physical stability and dissolution performance of solid dispersions is considered. The aim is to use correlations between drug molecular structure and solid dispersion performance to develop a platform to rapidly assess whether drug compounds will have favourable properties when formulated as a solid dispersion. Amorphous felodipine/copovidone solid dispersions are used as a model system to develop a suitable testing regime with regards to physical stability and dissolution performance. A laser light scattering technique developed in this work shows that morphological changes in felodipine/copovidone films exposed to water are due to polymer swelling. A combination of dissolution testing methodologies is also used to suggest a mechanism for the dissolution of bulk solid dispersion samples. Contributions of individual functional groups in the felodipine analogues to the physical stability and dissolution performance of their amorphous solid dispersions are assessed. Blocking of the felodipine amine hydrogen-bond-donor with an N-methyl, and the removal of chlorine substituents are both shown to reduce the physical stability of the solid dispersions. Correlations between molecular descriptors and data from the above experiments show that drug compounds are more likely to crystallise from solid dispersions with copovidone if they have a low log P, low relative molecular mass and low polarizability. Such correlations can form the basis of a screening method for the molecular design of analogous drug compounds likely to form high-performance solid dispersions with copovidone.

The potential application of inkjet printing technology to produce precisely dosage care is demonstrated in this thesis. Inkjet printing technology as it offers the opportunity to deliver quantities with high accuracy can produce medicines tailored for each patient. The viability of this method was first demonstrated by using Felodipine as an active pharmaceutical ingredient polyvinyl pirrolidone (PVP) as an excipient. Felodipine is an antihypertensive drug which is poorly soluble in water and PVP is a highly soluble polymer commonly used to improve drugs' bioavailability. These were dissolved at various ratios in a mixture of ethanol and DMSO (95/5). Using a piezoelectric driven dispenser, picolitre size droplets of the solutions were dispensed onto suitable hydrophobic substrates. The dried products were characterized using AFM, localized nano-thermal analysis and high resolution vibrational spectroscopy (ATR-IR and Raman). Results indicate intimate mixing of the micro-dot API and excipient mixtures. Specifically, ATR-IR confirmed the interaction of felodipine and PVP by means of hydrogen bonding. Nanothermal analysis indicates a single glass transition point which is lowered as the API concentration increases. Finally, confocal Raman microscopy mapping on single droplets allows the visualization of the homogeneous distribution of the drug. Also, capozide has been used as a model therapeutic system which could be produced rapidly as a viable formulation using the inkjet printing technology. Capozide consists of captopril, an angiotensin converting enzyme (ACE) inhibitor and hydrochlorothiazide, a thiazide diuretic drug, in varying ratios. These active pharmaceutical ingredients (APIs) and poly(lactic-co-glycolic acid) (PLGA) were dissolved in appropriate solvents and using a piezoelectric driven dispenser and pipetting, picolitre and microlitre size droplets respectively were deposited onto hydrophobic coated glass slides. Captopril and PLGA were dissolved in chloroform, ethanol and DMSO (75/18/7). Hydrochlorothiazide (HCT) and PLGA were dissolved in acetone and DMSO (93/7). The dried products where characterised using AFM and high resolution Raman microscopy. The results showed that both capropril and HCT are phase separated with the PLGA. Also, the dissolution profiles of the final products were measured using HPLC where it has been shown that PLGA can control the release of the drug from the formulation. These results are a promising first step to produce pharmaceutical by means of inkjet printing.

Most drugs have a considerably low solubility in the environmentally friendly hydrofluoroalkane propellants (HFAs) currently used in pressurized metered dose inhalers (pMDIs). As a consequence, instability can occur from crystal growth and Ostwald ripening of the system altering the therapeutic performance of the pMDI. Understanding and being able to predict the behaviour of such drugs in the propellant will help in selecting the correct co-solvents and/or surfactants to increase the stability of such formulations. When anhydrous beclomethasone dipropionate (BDP) is suspended in tricholoromonofluoromethane (CFC-11) rapid crystal growth occurs, leading to the formation of a clathrate. Since chlorofluorocarbon (CFCs) propellants have been replaced by HFAs, many questions arose concerning the ability of BDP to form clathrates in the HFA and any stability issues that arise from such reformulation. Clathrates are crystalline compounds consisting of a lattice of one type of molecule that hosts a second type of “guest” molecule within its structure. Since the solid state chemistry can significantly alter the physical interactions within a suspension formulation, it is crucial to determine the most stable crystalline form in the presence of the propellant. Successful formation of BDP CFC-11 clathrates were observed in this work as well as positive outcomes in terms of reduction in the surface energy and the force of adhesion within a model pMDI formulation (even after processing i.e. size reduction). Following this, HFA-134a and 227-ae were selected to determine any potential clathrate formation and to monitor their stability within a pMDI formulation. The focus of this project was to determine the stability of BDP and budesonide in HFA propellants, as well as the appropriateness of each formulation for pMDI use. This project considered the potential use of complementary surface and solid-state analytical tools to provide fundamental understanding of clathrate formation and their physicochemical characteristics. A special interest in understanding the fundamentals behind the process of Ostwald ripening, a process that affects drug particle size and their related stability and hence ultimately dose consistency was also considered. Atomic force microscopy (AFM) was used in order to determine its applicability in studying Ostwald ripening and surface activity of the different APIs in model propellant. Furthermore, the effects of a range of parameters that included storage time, co-solvents and surfactants on Ostwald ripening were taken into account. The work presented in this thesis has demonstrated that the formation of a propellant clathrate is favourable for APIs that to improve formulation stability through a reduction in particle surface energy. However, isolation and full characterisation of such HFA clathrates remains challenging due to their decreased stability when removed from the high pressure media of the pMDI device. This thesis shows that a combination of co-solvent and surfactant provides an effective reduction in instabilities due to Ostwald ripening of the pMDI formulation and a better control of particle size of the APIs within the formulation. This work provides a platform for future formulation development for pMDIs.

The aim of this work was to analyse the surface of biomedically relevant polymers with a range of surface sensitive techniques both in the interest of improving our knowledge of such polymeric delivery systems and the techniques used. Specifically time of flight- secondary ion mass spectrometry (ToF-SIMS) was a focus of this work complemented by a range of supportive surface and bulk analytical techniques. The new technique of X-ray photoelectron spectroscopy (XPS) depth profiling of organics was scrutinised through its application to a thin film blend of poly(l-lactic acid) (PLA) and the analgesic codeine in Chapter 3. Surface depletion of drug was observed in these films and was quantified for the first time with XPS. A multilayer model was created containing alternating layers of the codeine/PLA blend and biodegradable hydroxypropyl methylcellulose (HPMC) to test the application of SIMS to such formulations described in Chapter 4. Codeine was found to diffuse into a HPMC layer below it but not above due to a solubilisation of the bottom HPMC layer by the chloroform allowing small mobile codeine molecules to penetrate the layer below where the larger PLA chains were unable to. Interface widths observed when casting HPMC above a codeine/PLA layer was far broader than those observed when reversing layer order. This observation suggests HPMC is more sensitive to ion beam induced damage effects than PLA. The detailed characterisation of protein drug loaded polymeric microspheres was undertaken described in Chapter 5 revealing the discontinuous presence of surfactant at the surface and allowed for inferences to be made as to how the production process could be amended to tailor desired attributes. The work describes the thorough characterisation of biomedically relevant polymers with an array of surface sensitive techniques in the interest of improving the future description of such increasingly important formulations.

The regulatory framework in which pharmaceutical companies have to work has changed significantly since the late 1990’s. The development and implementation of risk based approaches to processing pharmaceutical powders allows the pharmaceutical manufacturers the freedom to adopt real-time release for their products whist reducing the regulatory burden for both the statutory bodies and the manufacturers. This thesis has been a collaboration between Buck Systems and the University of Birmingham School of Chemical Engineering to evaluate and develop methods which would enhance the way in which Buck Systems can, in co-operation with their clients, enhance their understanding of how powder properties affect their products that are used in pharmaceutical manufacturing to better comply with the changes in the regulatory environment. To this end simple and quick screening methods for characterisation of customers’ powders with a view to identifying potential problems prior to blending tests have been developed to replace the current ad hoc approach. These include the use of tests that have been relied on historically as well as newer, more universal and robust techniques such as automated shear cells and powder rheometers. Detailed characterisation trials have shown where these techniques can be successfully applied and where their limitations lie. Further work has shown how powder systems can be better evaluated within the existing HAZOP framework. Specific evaluation of the hopper design methodology has resulted in the development of an expert system to enable the rapid sensitivity analysis of design options. In addition the limits of the hopper design method have been explored and some limitations identified where significant overdesign may occur. The evaluation of content uniformity in a laboratory scale blender using specialist Positron Imaging equipment available at the University of Birmingham has also been undertaken. The unique study of the blender contents using Positron Emission Tomography has provided a range of insights into the way binary and ternary powder systems interdisperse.

The thesis describes the development and use of ultra-fast MRI techniques to quantitatively characterise the dissolution process of controlled drug release dosage forms. Implementations and validations of two quantitative single shot RARE based magnetic resonance imaging (MRI) protocols are described. Quantitative T2 (spin-spin relaxation time constant) and diffusion weighted single shot RARE images, both with acquisition time of less than 3 minutes, were achieved by preconditioning the standard RARE sequence with a hard pulse CPMG echo train or an alternating phase bipolar pulsed field gradient spin echo (APGSTE) diffusion sequence respectively. T2-preconditoined RARE and diffusion-preconditioned RARE experiments were carried out on 5 phantoms with T2 values between 29 – 2200 ms. The optimisation of the phase encoding start value (PESV) parameter in the MRI pulse sequence was carried out, and four centric phase encoding schemes were also investigated. Phantoms imaged using the conventional RARE phase encoding scheme under the optimal PESV value of -0.2, showed a percentage error difference of 11 % for absolute water concentration (Cwat) maps, and an error of 2 % in T2. Similarly an error of less than 2 % in the molecular self diffusion coefficient (D) was found compared to the corresponding reference value. The two quantitative preconditioned RARE pulse sequences were then used to follow the dissolution of hydroxypropylmethyl cellulose (HPMC) in both static and flowing environments. Finally, a case study was carried out on a coated tablet for the purpose of examining tablet coating efficiency upon scale up, where a pilot scale coated tablet was compared to a lab scale coated tablet. Two quite different dissolution behaviours of the two types of tablets were observed.

The STAT3 transcription factor plays a key role in a wide range of biological responses for cell survival and growth. Furthermore, the STAT3 signalling pathway has been found to be up-regulated in more than 70% of human tumours. To date STAT3 is a particularly promising molecular target for chemotherapeutic intervention, and a number of strategies are under investigation to selectively down-regulate STAT3 signalling in cancer cells to inhibit cell proliferation and promote cell death. In the molecularly-targeted drug discovery era, protein- protein interactions (PPIs) are emerging as an attractive class of novel targets. Proteins are associated with unique recognition patterns, thus targeting PPIs has the potential to develop highly selective drugs. In this context, blockade of STAT3 signalling through the modulation or inhibition of key protein-protein interactions is a valuable approach to inhibit STAT3 transcriptional activity. This research project has focused on the design and synthesis of small-molecule inhibitors of the STAT3:STAT3 protein interaction as a potential means to discover novel therapeutic agents using computational approaches such as virtual screening and structure-based design. In particular, a medicinal chemistry approach has been used to produce a novel library of potential PPI inhibitors based on a "hit" from an in silico screen. This library was screened using a primary PPI binding assay based on fluorescence polarisation (FP). Hits from this assay were screened in a MTS cell viability assay, a Trypan blue exclusion assay and a Luciferase reporter assay in STAT3-dependent (MDA-MB-231) and STAT3-null (A4) cell lines. Compounds with interesting activity in these assays were further studied in cellular assays to assess the extent of activity and specificity towards unphosphorylated STAT3, phosphorylated STAT3, phosphorylated STAT1, unphosphorylated STAT1 and the downstream mediators (i.e., Survivin, Bcl-XL and Cyclin D1) of STAT3 signalling. One compound identified, 60, has the ability to down-regulate IL-6 signalling at EC50 of 15 μM.