Dissolution testing is a tool towards prediction of dosage form behaviour under physiologically relevant conditions. The use of simple aqueous media dictated by the pharmacopoeias cannot predict the drug’s in vivo response as their physicochemical properties differ significantly from the complex environment of the gastrointestinal tract. To improve the predictive potential of drug dissolution, the development of more “biorelevant” media is essential. In particular, simulating drug dissolution in the gastric environment after administration of a meal still remains a challenge. Furthermore, except for the optimisation of the medium composition, the analysis of these usually complex heterogeneous media has also been challenging, due to the lack of a unified guideline for the selection of medium and analytical assay. The principal aim of the thesis was the development of a simple and robust analytical methodology, optimised on the basis of the drug’s physicochemical properties, interaction with excipients in a formulation and fat partitioning behaviour. The potential use of sample clean-up techniques including protein precipitation (PP) and solid phase extraction (SPE) was investigated. Optimised clean-up protocols were successfully used for extraction and quantification of drugs of a wide range of lipophilicity in milk-based fed biorelevant media. It was demonstrated that prior knowledge of the active ingredient’s physicochemical properties, such as log P, aqueous solubility, ionisation and protein binding can be used towards the selection of optimum extraction conditions. Moreover, the presence of certain excipients, when mixed with the APIs can significantly affect the methods’ efficiency and must thereforebe taken into consideration during analytical method development. Interactions between active ingredients and lipid part of the fed gastric content were also investigated, via development of biphasic “drug partition to fat” in vitro setups. The rate of drug partition to fat was successfully predicted based on the drug’s physicochemical properties and in vivo food effect. Finally, a simpler medium, alternative to Fed State Simulated Gastric Fluid (FeSSGF) was developed, requiring a less laborious extraction protocol. Overall, this thesis has provided useful insight on the critical aspects of fed gastric medium and analytical methodology development. It provides a point of reference for future work on better understanding on drug solubilisation in the gastric fed state and correlation with in vivo food effect.

Background. Response to anti-tumour necrosis factor (anti-TNF) therapy in patients with rheumatoid arthritis (RA) varies between patients. Incidence of sustained remission in the UK is not known, and factors contributing to its achievement are poorly understood. Prior knowledge of response would enable better targeting of anti-TNF therapy, leading to better outcomes and reduced morbidity. Aims. This thesis aims to identify incidence of sustained remission and low disease activity (LDA) in patients with rheumatoid arthritis (RA) taking anti-TNF therapy. Clinical and demographic factors associated with sustained remission and LDA were identified. Methods. I undertook a systematic literature review of the incidence of, and factors associated with, sustained remission in patients with RA taking anti-TNF therapy. Results informed a subsequent analysis of data extracted from the British Society for Rheumatology Biologics Register for Rheumatoid Arthritis (BSRBR-RA). I used two approaches to examine sustained remission and LDA. Firstly, pre-defined DAS28 thresholds were used to identify individuals in sustained remission and LDA. Secondly, a data-driven approach used latent class mixed modelling (LCMM) to identify independent trajectories of response within the data. Results. Sustained remission and LDA occurred infrequently in the literature review (range 4.2 – 38.1% sustained remission) and was uncommon in the BSRBR-RA (14.9% and 26.3% respectively), but had improved significantly over time. Significant associations were identified between the candidate variables and sustained remission and LDA, both using pre-defined thresholds and LCMM analyses. LCMM analyses identified response at six months to be a good indicator of long-term outcomes. Conclusions. Sustained remission and LDA remains uncommon, although outcomes are improving. Clinical and demographic features are associated with achieving these outcomes, suggesting it may be possible to use phenotypic features to guide therapy. Additionally, the clear response trajectories identified at six months, suggest it may be possible to identify non-responders to anti-TNF therapy earlier than six months.

Intra-articular (IA) administration is used for the symptomatic treatment of disorders such as Osteoarthritis (OA) and Rheumatoid Arthritis (RA) providing a local effect while avoiding systemic side effects. For appropriate IA formulations to be developed and for quality control purposes, appropriate dissolution models have significant importance. Developing these models poses a great challenge, while at the moment, there is no regulatory approved standard methods for testing drug dissolution from parenteral formulations. The principal aim of this thesis is the development of appropriate in-vitro compendial and biorelevant dissolution models of drug absorption after IA administration. A vast number of dissolution methods were investigated, based on their potential and suitability as compendial methods (USP apparatus I, II, III and IV, dialysis methods and bi-phasic models) and their applicability in simulating in- vivo conditions for the biorelevant dissolution aspect (Side-Bi-Side diffusion cells and bi- phasic models). Various parameters of the setups were successfully evaluated towards their effect in drug dissolution. The discrimination ability of the USP apparatus IV was demonstrated showing the significant potential of this method in order to be considered for compendial testing. For the biorelevant dissolution testing, biorelevant synovial fluids (BSFs) of healthy and disease states were developed according to performed in-vivo studies of physicochemical properties and solubility values of Triamcinolone Acetonide (TA). These media were also used in biorelevant dissolution testing, showing the ability of the tested systems to discriminate between these media. Finally, surface dissolution imaging has permitted direct visualisation of the solvation and dissolution of TA with a detailed insight in the characterisation of the dissolution process by evaluating the effect of surfactants and increased viscosity of the medium. Overall, this thesis provides essential information on the potential of dissolution models for being the primary method upon which the drug dissolution of IA formulations may be established.

Heme is an essential molecule for all aerobic life. It acts as a co-factor for a wide variety of different proteins involved in functions as diverse as oxygen transport, energy generation and catalysis. Free or unbound heme also has a less well understood role as a signalling molecule, involved in transcriptional control, protein degradation, circadian regulation and cellular redox homeostasis. However, free heme is also cytotoxic through its pro-oxidant activity and its ability to trigger inflammatory cascades. Investigations into the role of heme in physiological processes and disease have been hampered by the lack of specific heme sensors that can be used in live cells to distinguish the low concentrations of the free heme pool from the protein-bound fraction. In this thesis, potential heme sensors have been designed and synthesised based on the amino acid sequences of heme-binding regions in the proteins Bach1 and hemopexin. Synthetic peptides with a CP motif derived from Bach1 were found to bind heme in a 1:1 ratio with low micromolar affinity by UV-vis titrations. One such peptide was modified to incorporate a 7-azatryptophan residue in place of tryptophan and quenching of the red-shifted fluorescence was shown to be proportional to hemin concentration. This lead Bach1-derived peptide was used to measure heme levels in skin cell lysates and confirmed an increase in heme levels after treatment of cells with hemin or UVA irradiation. Further developments of this prototype sensor molecule have also been explored including the incorporation of a cell penetrating peptide sequence that improved uptake of the peptide by human skin cells in vitro. Conjugation of a second independent fluorophore to the heme-binding peptide was also investigated to prepare it for use in live cells, but this reduced the fluorescence of 7-azatryptophan, indicating that further optimisation of the fluorophore combination will ultimately be required. In summary, these studies provide the basis for the development of flexible peptide-based heme sensors that can be used to monitor heme levels in biological media. Further development should provide effective tools for probing the diverse physiological functions of heme, as well as aiding our understanding of how proteins such as Bach1 regulate the transcription of genes associated with heme degradation.

Drug-induced nephrotoxicity is a dose limiting factor and a common side effect of many drugs such as antibiotics, cancer chemotherapeutics or diagnostic agents and represents 20-25% of all cases of acute kidney injury (AKI). Due to a lack of representative animal models and metabolically competent renal cell lines, only 10.5% of the drug-induced nephrotoxicity cases can be predicted in preclinical studies. Furthermore, it has been recently accepted that mitochondria represent an important target for a broad spectrum of renal toxins. However, the supraphysiological glucose concentrations used in many culture conditions favour an energetic metabolic change known as the Crabtree effect, based on using glycolysis as the main route for ATP production and hence producing cell lines highly resistant to mitochondrial impairment. Therefore, there is a need for better physiologically-based renal in vitro models that can better assess drug-induced nephrotoxicity. Conditionally immortalised proximal tubule epithelial cells (ciPTECs) had been previously postulated as a novel renal model with good transporter expression that could produce reliable nephrotoxicity data for pharmacological studies. In order to confirm the applicability of the received ciPTECs for further drug transporter and toxicity studies, the transporter expression was assessed and compared with other cell lines such as HEK 293 cells as well as isolated cells from a proximal tubule (PT) fraction. The functionality of some transporters that are crucial in drug-induced nephrotoxicity such as megalin, MRP2, MRP4 and P-gp were assessed via CMFDA accumulation. Data revealed a very close mRNA expression of ciPTECs compared to the PT fraction and better transporter functionality than HEK 293 cells. Therefore, it was confirmed that ciPTECs hold the necessary transporter functionality to be a valuable nephrotoxicity model. The Crabtree effect had been previously circumvented in other cell lines by replacing glucose for galactose in the medium for 8 weeks. As a result, cells became highly aerobically dependant and more susceptible to mitotoxins. A recently developed strategy with only a 4 h pre-incubation period in galactose conditions was implemented in ciPTECs. As hypothesised, ciPTECs cultured in galactose medium displayed a significant upregulation of mitochondrial OXPHOS compared to glucose-grown cells, which caused a higher susceptibility in front of mitotoxins (rotenone EC50ATPglu / EC50ATPgal > 3125, p < 0.001 and antimycin A EC50ATPglu / EC50ATPgal > 14925, p < 0.001). Furthermore, when exposed to adefovir, a postulated mitotoxin in the clinic, galactose-cultured ciPTECs showed a significantly lower EC50 (EC50ATPglu / EC50 ATPgal > 6.12, p = 0.0049, 3 days). Tenofovir (TFV), a lower toxic analogue of adefovir, was also submitted to the glucose vs. galactose model ATP depletion did not reach 50%. Causes of mitochondrial dysfunction are variable but they all eventually trigger a perturbation of the respiratory parameters. Hence, to further explore the molecular mechanisms behind adefovir and TFV mitochondrial toxicity, ciPTECs bioenergetics were interrogated. After a 24 h adefovir exposure, a decrease in oxygen consumption linked to ATP production was detected as well as a 40% reduction of the coupling efficiency at 100 μM. Electron transport chain (ETC) complexes were also quantified and results revealed a depletion of complex I (45%) and complex IV (20%). These are the only two complexes with an important mtDNA encoded core. Furthermore, reactive oxygen species (ROS) levels increased 2 fold after adefovir exposure (48 h). In conclusion, these data evidenced that adefovir targets either mitochondrial RNA or protein synthesis, impeding a correct protein assembling of ETC complexes and therefore hampering cell respiration. However, other factors such as ROS might also play an important role. No decrease in oxygen consumption was detected after ciPTECs were exposed to TFV for 15 days. In contrast, a dose-dependent increase in extracellular acidification rate (ECAR) was observed, which could be an initial sign of mitochondrial dysfunction. Although ciPTECs exhibit most of the necessary features to investigate nephrotoxicity such as good transporter expression, they cannot take into account different people's pharmacokinetics or transporter polymorphisms that alter drug-induced toxicity. In contrast, urine-derived progenitor cells (UPCs) represent a non-invasive and limitless source of kidney cells that could be differentiated into PT cells. Even though the PT-differentiation was not fully achieved, we successfully isolated three colonies of UPCs that reached passaged 10 with similar clonogenicity values to cancer cell lines (31%) at the exponential growth phase. Therefore, if the differentiation process could be optimised, urine-derived cells could have the potential to be used as a patient-specific nephrotoxicity model as well as being applied in personalised medicine and regenerative studies. In summary, the data presented in this thesis demonstrate that drug-induced mitochondrial dysfunction responsible for nephrotoxic events can be detected using the ciPTECs glucose vs. galactose in vitro model. Furthermore, in addition of screening for mitotoxic compounds, it has been shown that the model can also be used as a tool to investigate the molecular events preceding adefovir toxicity as well as other undisclosed mitotoxicities. Finally, the work undertaken has also demonstrated that UPCs and PT-differentiated UPCs hold promising answers to tackle and prevent drug-induced nephrotoxicity.

Motor neurone disease refers to a group of neurological disorders that result from progressive degeneration of the motor neurones leading to death from respiratory failure within 3-5 years from the onset of symptoms. Amyotrophic lateral sclerosis (ALS) is one of the most common forms of motor neurone disease, and can be described as entailing the involvement of both upper and lower motor neurones. Usually, the disease is obvious and presents as asymmetrical weakness in the limbs and progressive muscular atrophy. Over the last two decades more than 30 genes have been identified as involved in ALS pathology. These include PARK7 and FUS (Fused in sarcoma) which are the subject of this thesis. FUS is a multifunctional protein that has ubiquitous expression and is involved in different steps of RNA processing such as mRNA and miRNA. 10% of ALS cases are heritable and mutation of the FUS gene is found in 3-5% of familial forms of ALS. Therefore the FUS gene is important for it association as a candidate gene that is postulated to be important for ALS, in addition to various types of cancer. Mutations with FUS gene have been found in autopsy samples from the brain and spinal cord of patients with ALS. Understanding the regulation of FUS gene expression may, therefore, give insights into how its stimulus inducible expression may be associated with neurological disorders. My hypothesis is that the evolutionary conserved regions (ECRs) and primate specific retrotransposons of the SINE-VNTR Alu (SVA) are regulatory domains from the human FUS gene and PARK7 genes. Consequently, the aim of this thesis was to develop an in vivo model to validate their regulation. Comparative genomic analysis was used between distant species, utilizing the ECR browser and UCSC browser to identify conserved regions from the FUS gene. It is demonstrated that ECR and SVA, xviii which can drive reporter gene activity in vitro (in the neuroblastoma cell line), are also capable of driving reporter gene expression in the chick embryo neural tube and brain at embryonic day 5. In conclusion, these identified important ECRs from human FUS gene act as regulatory domains. In addition, the SVAs, representing the most recent retrotransposon to enter the human genome also was showed to have regulatory properties in FUS and PARK7 genes. Furthermore, the thesis demonstrates that these elements regulate gene expression in vitro and in vivo. This supports the idea that employment of the chick embryo as a useful and informative model system to analyse mammalian gene regulation.

Nosocomial bloodstream infection (BSI) is the most common type of hospital-acquired infection in paediatric patients and a major cause of morbidity and mortality worldwide. Methicillin-resistant staphylococci (CoNS and MRSA) are a leading cause of hospital-acquired neonatal sepsis and BSI. Glycopeptides (vancomycin and teicoplanin) constitute the current mainstay of therapy. There is limited antimicrobial PK-PD data available for neonates and children and optimal drug exposures resulting in maximal efficacy and suppression of resistance are not known. A translational pharmacological approach can be used to build the evidence required to optimize the current use of antimicrobial therapy in children. Pre-clinical experimental (in vitro and in vivo) and clinical PK-PD work was conducted throughout this thesis to improve our understanding of the PK-PD relationships of vancomycin and teicoplanin against CoNS and MRSA. The in vitro HFIM defined the relevant PD indexes and free drug exposures associated with maximal bacterial killing and suppression of resistance. The in vivo models (a rabbit central-line associated BSI and a mouse neutropenic thigh infection model) validated the in vitro findings. CRP concentrations were incorporated as an in vivo PD input. A clinical PK study of teicoplanin in 57 patients (neonates, infants and older children) was conducted and the population PK parameters estimated. PK-PD modelling techniques were used to analyse the PK-PD data and bridge the experimental results to human patients by means of Monte Carlo (MC) simulations. Vancomycin and teicoplanin displayed a concentration-dependent pattern of activity. An AUC:MIC ratio was associated with maximal antibacterial activity and suppression of resistance. Based on MC simulations, the probability of the in vivo target attainment with currently used teicoplanin dosage regimens results insufficient to treat a majority of patients with MRSA infection. High teicoplanin PK variability was identified in children. Weight, age and renal function were the best explanatory covariates of PK variability. Wider drug exposure distribution is observed in the paediatric population with respect to adults. A patient-tailored TDM approach with the aid of a Bayesian feedback adaptive control tool is required to ensure individual patients achieve optimal drug exposures in a precise and safe manner. The defined pre-clinical optimal targets of exposure for vancomycin-CoNS and teicoplanin-MRSA now need to be prospectively evaluated in patients. Currently used teicoplanin dosage regimens in both, adults and children, may be insufficient to treat a high proportion of patients with serious MRSA infection. Current EUCAST clinical breakpoint may need to be revised for teicoplanin against MRSA. The current strategy of using teicoplanin fixed population-based antibiotic regimens results in a wide range of drug exposures in neonates and children. An individualised dosing and TDM approach can ensure optimal target attainment at the individual level and in real-time.

The understanding of the mechanism, by which bradykinin (BK) effects ureteric contractility, is limited. The primary aim of this study was to determine the expression and the distribution of B2 receptors along the length of the ureter and study the effects of BK on Ca2+ signalling and contractile activity of the rat ureteric smooth muscle. Both the Ca2+-dependent and Ca2+-independent pathways, underlying the stimulant action of BK on the ureteric smooth muscle, have been investigated. Immunohistochemistry was used to identify the presence and the distribution of B2 receptors along the ureter. Confocal imaging combined with force measurements under different physiological conditions was used to study the effects of BK on Ca2+ signalling and force of the ureteric smooth muscle in situ. The effect of the selective blocker of Rho-associated kinase, Y-27632, on force/ Ca2+ relationship was investigated. It was found that the distribution of the B2 receptors is non-homologous along the length of the rat ureter, with the expression being greater in the upper ureter and lowest in the middle and the lower ureter. It was found that BK caused a complex contractile response, consisting of an initial phasic component, associated with the Ca2+ release from the store, which appeared as asynchronous Ca2+ waves, followed by a sustained component, associated with Ca2+ entry via store/receptor operated Ca2+ channels that was superimposed by multiple phasic contractions, associated with propagating intercellular Ca2+ waves. The L-type Ca2+ channel blocker, Nifedipine, selectively blocked the intercellular Ca2+ wave and the repeated phasic contraction, but had no effect on the initial phasic contraction, associated with the Ca2+ release from the sarcoplasmic reticulum. Nifedipine, also had no effect on the sustained slow component, associated with the Ca2+ entry via store/receptor operated Ca2+ channels. Y-27632 had no effects on the Ca2+ signalling, associated with the Ca2+ release from the store or the Ca2+ entry via store/receptor operated Ca2+ channels, but had significant inhibitory effects on the initial phasic and sustained tonic component of BK-induced contractile response. There was a significant reduction in the amplitude and an increase in the rate of relaxation of the initial phasic component, suggesting an increase of the activity of myosin light chain phosphatase, responsible for dephosphorylation of MLC20 and relaxation of force in the smooth muscles. Collectively, the data obtained indicate that both Ca2+-dependent and Ca2+-independent mechanisms are involved in BK-induced stimulant action on the rat ureteric smooth muscle.

The complement system provides an essential role in the orchestration of an effective immune response against microbial infection. Activation leads to the generation of C5a which activates receptors expressed on immune cells promoting microbial clearance. Dysregulation of the complement system that results in the excessive production of C5a can lead to host tissue damage. This has fuelled drug discovery efforts to therapeutically block the actions of C5a to treat diseases associated with dysregulation of the complement system. C5a is rapidly degraded to C5a des Arg, a mechanism which is considered to inactivate its effector functions, however, there is incomplete understanding of precise functions of C5a and C5a des Arg on the cognate C5a receptors, C5a1 and C5a2. The research conducted here attempted to improve upon this understanding as well as fuel future drug discovery efforts that target this component of the complement system. Functional assays using human isolated neutrophils or engineered cell lines along with C5a1 and C5a2 receptor selective antagonists were employed to assess the contribution of each C5a receptor to functional responses elicited by human purified C5a and C5a des Arg. For each functional endpoint investigated, the relative activity of C5a des Arg was compared with C5a using the Log(Emax/EC50) transformation. Single point mutagenesis was performed on the C5a1 receptor to relate differences in agonist functional activity to mode of agonist binding. The data presented here supports the hypothesis that C5a des Arg behaves as a biased agonist in relation to C5a. Via the C5a1 receptor, C5a des Arg produces neutrophil phenotypes that are involved in the orchestration of immune cell recruitment to sites of infection. However, unlike C5a, C5a des Arg does not induce a respiratory burst response that leads to the generation of hypochlorous acid. The biased agonism of C5a des Arg appears to be brought about by its inability to interact with amino acid residues within the seventh transmembrane domain of the C5a1 receptor, which prevents the receptor recruitment of -arrestins. Furthermore, data presented here also show that the C5a2 receptor does not directly contribute to C5a or C5a des Arg mediated activation of the human isolated neutrophil. Taken together, the data support a hypothesis whereby the generation of C5a des Arg, through C5a1 receptor, limits the ability of C5a to promote the production of potentially damaging respiratory burst while still enabling immune cell extravasation.

No description available.