Anticipation of drug-drug interactions (DDIs) in the paediatric population are merely based on data generated in adults. Hence decision on avoiding certain combinations or attempts to adjust and manage the doses under combination-therapy are mainly speculative from the knowledge of what occurs in adults. However, due to developmental changes in elimination pathways from birth to adolescents, the assumption of DDIs being similar in adults and children might not be correct. This thesis firstly identifies and quantitatively compares the reported DDIs in paediatric and adult populations through a systematic literature review of DDIs reported in paediatric subjects. The study highlights the clear paucity of the data in children younger than 2 years. Therefore, the logical approach to test this hypothesis has been through modelling and simulation and incorporation of the biological knowledge on ontogeny of various enzymes and other elimination routes. The magnitude of any metabolic DDI depends on fractional importance of inhibited pathway which may not necessarily be the same in young children when compared to adults. To show this disparity between rate of ontogeny for metabolic pathways, the ontogeny pattern of CYP enzymes and renal function were analysed systematically. Bootstrap methodology was used to account for variability, and to define the age range over which a statistical difference is likely between each pair of specific pathways. A number of DDIs were simulated for virtual compounds to highlight the possibility that the magnitude of DDI can be influenced by age. Depending on the extent of contribution of metabolic pathways, neonates could be more sensitive to DDI than adults in certain scenarios or vice versa. Thus, extrapolation from adult DDI data may not be applicable across paediatric age groups. The uncertainty around the ontogeny functions based on in vitro information led us to carry out comprehensive performance verification for in vivo data on probe substrates of CYP1A2, -2C9 and 3A4 and assess the predictions of clearance (CL) by monitoring AUC. Although the evaluation showed that in most cases predictions were within two fold of observed data in adult and paediatric studies, the outcome suggests that the current ontogeny profiles result in under-prediction of CL values compared to clinical studies in infants and children and there is a need for better ontogeny models. Therefore, we derived novel ontogeny functions for CYP1A2 and CYP3A based on caffeine-theophylline and midazolam in vivo data. Age related CL data for caffeine, theophylline and midazolam were reconstructed back to intrinsic CL values per milligram of microsomal protein and best fit ontogeny models for CYP1A2 and CYP3A were derived from these data. The function for CYP1A2 describes an increase in relative intrinsic metabolic CL from birth to 3 years followed by a decrease to adult values. The function for CYP3A4 describes a continuous rise in relative intrinsic metabolic CL, reaching the adult value at about 2 years of age. The new models were validated by showing improved predictions of the systemic CL of ropivacaine (major CYP1A2 substrate; minor CYP3A4 substrate) and alfentanil (major CYP3A4 substrate) compared to those using a previous ontogeny function based on in vitro data. When implementing enzyme ontogeny functions it is important to consider potential confounding factors related to disease, anaesthesia and surgery that may affect the prediction of net in vivo CL. Finally, we demonstrated the application of paediatric physiologically-based pharmacokinetic (p-PBPK) models for calculation of sample size in paediatric clinical pharmacokinetic (PK) studies in a methodology suggested by Wang et al., based on desired precision for a PK parameter of interest. We obtained estimates of variability for CL, volume of distribution and area under the plasma concentration-time curve for 5 different drugs from (i) adult and paediatric classic clinical PK studies, and (ii) p-PBPK combined with in vitro-in vivo extrapolation. The estimates were applied to the sample size calculation proposal methodology for non-compartmental analysis. There were clear and drug dependent differences in calculated sample size based on various estimates of variability and overall, there was no consistent discrepancy in the sample size calculated according to the source of variability used for sample size calculations. The results are discussed in terms of their potential impact on the clinical PK studies in children. In general, considering the sensitivity of paediatric clinical PK studies and paucity of data in this group of patients, the use of p-PBPK models may offer an interim solution to uncovering age bands with potential higher vulnerability to DDI. However, these models require further refinements and testing before widely used in clinical practice with confidence.

Rheumatoid arthritis is an auto-immune inflammatory disease that leads to stiff and swollen joints. Patients also have severe bone destruction of the affected joints and another common symptom of rheumatoid arthritis is a generalised bone loss that can lead to osteoporosis. Currently, there are many treatments for rheumatoid arthritis, which provide a recession of the inflammatory symptoms. However, none of these treatments are able to provide a complete protection against the rheumatoid arthritis-induced bone loss. Furthermore, the most effective available treatments such as glucocorticoids or the new biological drugs are not optimal since they either cause severe side effects or are very expensive and difficult to produce. Hence, there is a real need for new cost-effective treatments that can act on both inflammation and bone loss symptoms of rheumatoid arthritis. ABD compounds are small molecules, relatively easy to synthesize at reasonable cost. In this thesis, I discuss the effects of these small molecules on both rheumatoid arthritis-induced inflammation and bone loss. Daily treatments with the ketones ABD328 and ABD345, or with the sulphonamide ABD455 prevent inflammation in an animal model of rheumatoid arthritis. Furthermore, micro-CT and histology analysis showed that these treatments also provide a reliable protection against bone destruction of affected joints and generalised bone loss. In vitro data showed that this protective effect on bone was osteoclast specific. Indeed, Ishow here that treatment of other bone cells (such as osteoblasts or macrophages) with ABD compounds does not affect their biology. The mechanism of action of these compounds has also been studied and I show here that ABD compounds inhibit both inflammation and osteoclastogenesis by inhibiting the signalling pathways that are activated in response to pro-inflammatory cytokines such as TNF . This work led to the design and synthesis of further improved compounds, such as ABD599, that are currently considered as very interesting candidates for clinical trials. In conclusion, the ABD compounds, as small cost-effective molecules, represent a novel class of rheumatoid arthritis treatments by acting on both inflammation and bone loss symptoms of the disease.

The cannabinoid receptors CB1 and CB2, which belong to the rhodopsin family of GPCRs, are implicated in the pathology of various disease states. As drugs targeting these receptors remain limited, novel cannabinoid receptor ligands represent an unmet need with substantial therapeutic potential. We present here the construction and application of homology models of the human CB1 and CB2 cannabinoid receptors based on the crystal structure of the human adenosine A2A receptor for the structure-based design of novel ligands based on the fenofibrate scaffold. Models were refined through molecular dynamic simulations in a lipid bilayer, and were validated via the prediction of known ligand binding affinities, enrichment studies and assessment of predicted ligand binding modes. These validated models were subsequently used in predicting the binding mode of fenofibrate derivatives to the cannabinoid receptors. The predicted binding mode of these fenofibrate derivatives to the CB2 receptor showed good agreement with known mutagenesis data, indicating the binding of these compounds to be stabilized primarily by hydrogen bonds with W5.43 and C7.42, aromatic stacking with F2.57, F3.36 and W6.48, and hydrophobic contact with F2.64, V3.32 and I5.47. A series of novel ligands was derived based on these findings, docked into our model, synthesized and pharmacologically evaluated at the CB2 receptor. The pharmacology of these ligands validated our modelling predictions and binding mode hypothesis, with several of these ligands showing unique pharmacology by binding in an allosteric manner. These findings may be used to guide the design of further derivatives and highlight the promise of the fenofibrate scaffold in the development of novel CB2 receptor ligands.

This thesis initially describes the quest for sequence selective DNA followed by the search for new antimicrobial compounds. The first four chapters are based on DNA recognition with new cancer therapeutics being the long term goal, while the remaining three focus on finding new drugs for bacteria.

Polymer-based drug delivery systems have fantastic potential in chemotherapy as they can reduce drug side effects, help in patient compliance and provide targeting. Nanoprecipitation is used to encapsulate small drug molecules into polymer nanoparticles to form a drug delivery system. A major obstacle in polymer-based drug delivery systems reaching the clinic is their inability to load sufficient drug molecules. Little is known about the processes involved in the encapsulation of drug molecules into these delivery systems. An insight into the processes that govern the formation of these particles and encapsulation of small drug molecules within them is therefore desirable. We used molecular dynamics to model nanoprecipitation by simulating the dispersion of an acetone drop, containing polymer, into water containing drug. To allow sufficient dispersion of acetone a large amount of water is required, thus coarse-graining becomes mandatory. However, we maintain accuracy for our polymer-drug interactions by using a multiscale force field. Atomistic polymer and drug molecules contain coarse-grain virtual sites which facilitate interactions with the coarse-grain solvent molecules. We also employed fully atomistic reference simulations via resolution transformation to optimise our multiscale force field. This thesis details the theory and design behind this model of nanoprecipitation including how other techniques produced inferior results. Initial simulations with our multiscale model matched an experimental trend and were shown to be accurate relative to atomistic reference simulations. We also analysed a fully atomistic simulation of nanoprecipitation that took several months to complete. This atomistic simulation was used as a reference to update the multiscale force field. The updated force field improved on some aspects of the simulation but there are still areas that need improvement. Insight from the simulations provides an understanding of the experimental results and trends. The transferability of the model should help in designing more efficient polymer-based drug delivery systems in the future. We conclude with future work on modelling polymer-based drug delivery systems including alternate methods to gain understanding of not only drug incorporation but also drug release.

Paracetamol is the most common drug taken in overdose in the UK. Although it has been used in overdose for about 50 years, there are many aspects of its toxicity and treatment that are not fully understood. In this thesis a series of related studies on paracetamol overdose are reported. The nephrotoxic effects of paracetamol in overdose have long been recognised. To better understand the mechanisms of this effect the effect of acute paracetamol overdose on plasma electrolytes were investigated, both retrospectively and, more intensively, prospectively. The results of these studies showed paracetamol overdose is associated with dose-related hypokalemia, and kaliuresis of short duration (<24h), suggesting a specific renal effect of paracetamol in overdose, perhaps via cyclo-oxygenase inhibition. This effect seems distinct from any nephrotoxic effect of paracetamol. In the third study the possible impact of features at admission, including renal impairment, on outcomes in a large cohort of patients who developed severe liver injury following paracetamol overdose was evaluated retrospectively. The key finding was that plasma creatinine, and gamma glutamyl transpeptidase, at first admission appeared to be useful predictors of poor outcome. The last three studies focus on antidote treatment of paracetamol overdose. Intravenous acetylcysteine (NAC) has been used as treatment of choice for over 30 years in patients who are at risk of hepatotoxicity. There are reports of liver failure and death in patients who have “non-toxic” plasma paracetamol concentrations on the UKL nomogram, and who are therefore not treated. To better understand this, the frequency of liver failure in patients who had low paracetamol was assessed by examining retrospective data from the Scottish Liver Unit over a 12-year period. Similar data was collected in the University of Newcastle upon Tyne by colleagues there. Only a small percentage of patients developed hepatotoxicity when initial paracetamol was low. It was concluded that on a cost-benefit basis the current thresholds for antidote treatment should not be lowered. The final 2 studies examine adverse reactions (ADRs) to NAC, a common clinical problem. The pattern and mechanisms of ADRs in man are not well described or understood. Factors influencing the frequency of adverse effects were studied in a prospective manner. Paracetamol concentration and male gender were protective and family history of allergy was a risk factor for adverse effects in this cohort. In a smaller focussed study the roles of histamine and other biomarkers as underlying pathophysiological mechanisms in ADR occurrence were studied. The severity of ADRs correlated with the extent of histamine release, which was independent of tryptase increase, suggesting a non-mast cell source. The mechanisms by which paracetamol might lessen histamine release require further investigation.

Thalidomide was discovered to be teratogenic over 50 years ago, but is far from being a historical problem. A new generation of thalidomide survivors have been reported in Brazil, where the drug is used to treat leprosy complications and multiple myeloma. The mechanisms underlying thalidomide teratogenesis are not fully understood. Cereblon (CRBN) protein has been identified as a target of thalidomide. Cereblon is part of an E3 ubiquitin ligase complex with Damaged DNA Binding protein 1 and Cullin-4A. I have analysed the expression patterns of CRBN and its binding partners in wildtype and thalidomide-treated chicken and zebrafish embryos. My results show that CRBN complex genes are weakly expressed in multiple tissues, including those not affected by thalidomide, and do not change following thalidomide exposure. I have also investigated the teratogenic potential of Primodos, a drug claimed to be “the forgotten thalidomide”. This drug was used as a pregnancy test between 1950's and 1970's. Primodos is alleged to be teratogenic but still is not recognised as one. Several epidemiological studies have been conducted, with conflicting results. I have been analysing the teratogenic properties of Primodos in chicken and zebrafish embryos and found that Primodos causes a range of malformations in zebrafish embryos. I have also carried out molecular analyses that show Primodos causes gene expression changes, changes in blood vessel patterning and neurite outgrowth in vivo and in vitro and increase in cell death. Finally, I have investigated the role of blood vessels in limb development and patterning. Using an antiangiogenic analogue of thalidomide, I found that inducing blood vessel loss in different regions of the forelimb bud of developing chicken results in different phenotypes. My results suggest that blood vessels might be involved in limb patterning and progress the understanding of limb defects observed in thalidomide survivors.

Staphylococcus epidermidis biofilm formation is a primary cause of medical device infections, which are persistent and difficult to eradicate because biofilms intrinsically exhibit a naturally high level of antibiotic resistance. Although biofilm antibiotic resistance or tolerance is a multifactorial process, some mechanisms such as limited diffusion, low metabolic activity and persister cells, contribute to the failure of antibiotics in the treatment of biofilm infections. Current, antibiotic treatment strategies may provide biofilm infections with intermittent exposure to sub-minimum inhibitory concentrations (sub-MIC) of antibiotics. Biofilms have been shown to display an increase in antibiotic tolerance when exposed to antibiotics at sub-MIC. Such mechanisms of adaptive antibiotic resistance are not well characterized but are of extreme clinical importance. This project showed that exposure to sub-MIC vancomycin increases the virulence of S. epidermidis biofilms because it induces vancomycin tolerance. BODIPY FL-vancomycin (fluorescent vancomycin conjugate) and confocal microscopy were used to show that the penetration of vancomycin through sub-MIC vancomycin pre-treated S. epidermidis biofilms was impeded, when compared to control, untreated biofilms. In addition, the results showed that a wide range of sub-MIC vancomycin concentrations induced an increased amount of extracellular DNA (eDNA) within the matrix of sub-MIC vancomycin treated biofilms. Finally, a set of ex vivo experiments using extracted exogenous S. epidermidis DNA revealed that exogenous S. epidermidis DNA binds vancomycin. Collectively these findings suggest that sub-MIC vancomycin exposure increase the abundance of eDNA in the matrix of S. epidermidis biofilms, which protects the biofilm community from subsequent vancomycin exposure by binding vancomycin as it travels through the matrix. Therefore the work in this project provides details of an eDNA-based mechanism of adaptive antibiotic tolerance in sub-MIC vancomycin treated S. epidermidis biofilms, which might be an important factor in the persistence of biofilms infections.

Conventional adult dosage forms such as tablets and capsules are often not suitable for the paediatric and geriatric population due to either swallowing difficulties or a requirement for tailored dosing to meet individual needs. Alternative oral formulations such as orally disintegrating tablets (ODTs) are available; however these usually require the incorporation of taste masking techniques. One approach to taste masking is to reduce contact between the bitter active pharmaceutical ingredient (API) and taste buds. This may be achieved by hindering release in the oral cavity using reverse enteric polymeric coatings. In vitro dissolution testing can be employed to elucidate taste masking capability by quantifying release of the API in simulated oral cavity conditions. This provides a robust analytical approach circumventing the expense and ethical challenges associated with human taste testing panels or animal testing. To achieve taste masking, drug release should be below the bitterness threshold concentration of the API. A vast array of dissolution methodologies has been employed in the evaluation of taste masked formulation performance in literature, with little agreement between approaches, and a lack of biorelevance. For optimal predictability, the dissolution test should be biorelevant and the dissolution media should mimic human saliva as closely as possible. Human saliva is thus a biological fluid of great importance in the field of dissolution testing. However, until now, no consensus has been reached on its key characteristics relevant to dissolution testing. As a result, it is difficult to select or develop an in vitro dissolution medium to best represent human saliva. In this thesis, for the first time, the pH, buffer capacity, surface tension, viscosity and flow rate of both unstimulated (US) and stimulated (SS) human saliva were investigated with a sufficient number of participants to generate statistically meaningful results (Chapter 3). This provides a platform of reference for future dissolution studies using simulated salivary fluids (SSFs).

Campylobacter is a major cause of acute bacterial gastroenteritis worldwide, with the highest number of infections being attributed to Campylobacter jejuni. C. jejuni is a Gram negative, spiral, motile bacterium that belongs to the campylobacterales order and is related to both Helicobacter spp. and Wolinella sp. It has long been established that proton pump inhibitors (PPIs) and other benzimidazole derivatives display anti-Helicobacter activity in vitro. PPIs have in the past been shown to affect Helicobacter pylori growth, survival, motility, morphology, adhesion/invasion potential and susceptibility to conventional antibiotics. PPIs are highly effective drugs that are well tolerated, safe for prolonged daily use and are therefore in high demand. Both the PPIs omeprazole and lansoprazole featured in the top ten drugs prescribed in England in 2014. In 2014 Campylobacter was also the most commonly diagnosed gastrointestinal infection in Scotland, in England and Wales and also in Europe. It has previously been generally accepted that patients who are being treated with PPIs are more susceptible to enteric infections such as Campylobacter than people not taking PPIs. The effect of PPI exposure on H. pylori has been investigated rigorously in the past. A single previous study has hinted that PPIs may also be capable of affecting the related organism C. jejuni,but investigations have been extremely limited in comparison to those investigating the effect of PPIs on H. pylori. This study has investigated the in vitro effects of direct contact with PPIs on the biology ofC. jejuni. Exposure to the PPI pantoprazole was found to affect C. jejuni growth/survival, motility, morphology, biofilm formation, invasion potential and susceptibility to some conventional antibiotics. Microarray studies showed that the cmeA and Cj0561c genes were significantly up-regulated in response to pantoprazole exposure and a CmeABC deficient mutant was found to be significantly more susceptible to killing by pantoprazole than was the parent strain. Proteomic analysis indicated that the oxidative stress response of C. jejuni was induced following exposure to sub-lethal concentrations of pantoprazole. C. jejuni gene expression was assessed using qRT-PCR and the genes encoding for thiol peroxidase and GroEL co-chaperonin (both involved in the C. jejuni oxidative stress response) were found to be around four times higher in response to exposure to sub-lethal concentrations of pantoprazole. Experiments using the oxidative stress inhibitors thiourea (a hydroxyl radical quencher) and bipyridyl (a ferrous iron chelator) showed that killing by pantoprazole was not mediated by hydroxyl radical production.