Synthetic targets that mimic key structural and conformational features in proteins are of intense scientific interest. These structures can reproduce the biological activity of their naturally occurring counterparts, while improving their properties in vivo. This project focuses on the synthesis of beta-turn mimetic structures, based on the opioid pentapeptide leu‑enkephalin. More specifically, the target mimetics in this project are based on the enkephalin derivative, leu‑enkephalinamide. Leu-enkephalin is conformationally very flexible, giving it the ability to bind to different opioid receptors. This results not only in the physiological relief of pain, but also in various potential side effects such as miosis or physical dependency. Natural peptide structures also have limited suitability for drug applications because of their poor transport properties and their tendency to undergo proteolytic degradation. The synthesis of macrocyclic mimetics can overcome these problems and give a conformationally rigid molecule that exhibits a desired biological activity. This thesis will discuss some published examples of beta-turn mimetic structures that have been designed and synthesised by research groups working in this field. The examples that are shown have been chosen to illustrate some of the work that has taken place in this area over recent years. Advances in the synthesis of ynamides and other ynamine analogues is also discussed, due to the importance of this type of chemistry in the research project. Efforts towards the synthesis of several 10-membered macrocycles are described herein. The target structures are model compounds that are designed to explore varied chemical approaches towards such ring systems. This thesis will discuss the synthesis of several linear precursors to the proposed macrocycles and efforts to cyclise these structures to form the desired targets. The targets that were synthesized employed varied approaches to the key cyclisation reaction. Initially, a metal-mediated coupling approach to an ynamide target was chosen. This was subsequently refined by including a proline residue in the linear precursor to bring the reacting groups into closer proximity. Finally, several approaches were investigated to close an alkyne bonded macrocycle by coupling the amide functionality at the opposite side of the ring.

Inter-individual variation in pharmacokinetic parameters of drugs can have profound effects on drug safety in children. Midazolam and morphine are among the most commonly used drugs in critically children. Theophylline has seen several cycles of enthusiasm and unpopularity over the years, although oral theophylline is now rarely used, IV aminophylline is still used regularly in severe asthma. These drugs are metabolised by hepatic enzymes (CYP3A4, CYP1A2 and glucuronidation) which have variable expression. Three systematic reviews were conducted in order to explore the inter-individual variation of clearance of these drugs in children. The first systematic review evaluated the inter-individual variability of midazolam clearance in children. Midazolam is predominantly metabolised by CYP3A4. Twenty two PK studies were identified. The mean clearance of midazolam varied between 0.78 to 3.5 ml/min/kg in neonates and 1.1 to 15 ml/min/kg in children. Age was a statistically significant predictor of clearance (p < 0.05). Critical illness was however not a statistically significant predictor of midazolam clearance after adjusting for other covariates (p=0.279). There was a statistically significant difference between the coefficient of variation of midazolam clearance in preterm neonates (91%) and children (40%) (p=0.002). However, there was no significant difference between the CV in critically ill and non-critically ill children. A second systematic review evaluated the variability of theophylline clearance. Theophylline is metabolised by CYP1A2. Twenty nine studies were identified. Mean clearance of theophylline varied between 0.2 and 2 ml/min/kg. Age was a significant predictor of theophylline clearance (p<0.05). There was, however, no significant difference between the CV of theophylline in any age group. The CV of theophylline clearance was not significantly different between critically ill (35%) and non-critically ill (39%) (p=0.403). A sub-analysis of children also did not show any significant difference between critically ill and non-critically ill (p=0.418). A third systematic review evaluated the variability of morphine clearance in children. Morphine is metabolised by UGT. Twenty studies were identified. The mean clearance of the studies identified varied between 2 and 16 ml/min/kg in neonates and 19 to 52 ml/min/kg in children. Critical illness was not a statistically significant predictor of morphine clearance. Analyses of the limited data showed no statistically significant differences in CV between any age groups. There was also no statistically significant difference between the CV in critically ill and non-critically ill children. In all the studies, a major limitation was the limited number of PK studies in children. Invasive studies should be avoided in children therefore, a final systematic review evaluated the invasiveness of PK studies over two decades. The number of blood samples collected per child was significantly lower in studies carried out between 2004-2014 than those between 1981-1990 (p=0.013). Furthermore, the total volume of blood collected in 24 hours for PK studies was significantly lower in new decade than old (p=0.025). However, there was no difference in the volume of blood collected per sample. There were 35 population PK studies, all of which were new studies. The median number of blood samples in population PK studies (median 6, [IQR: 4-9]) was significantly lower than non-population PK studies (median: 8, [IQR: 6-10]) (p=0.007). In conclusion, age is a risk factor for inter-individual variation of midazolam clearance in children. It is also an important predictor of midazolam, morphine and theophylline clearance in children. Therefore, age appropriate dosing is important. More PK studies are required to determine the effect of critical illness on the variability of clearance of these drugs. The utilisation of population PK methods should be encouraged to minimise invasiveness of PK studies. New methodologies for reducing sample volumes and frequency should be considered in all studies.

The glucocorticoid receptor (GR) is a member of the nuclear hormone receptors family and has been shown to exert significant effects on the induction of cytochrome P450 (CYP) enzymes responsible for the metabolism of many xenobiotics. CYP3A4/5 and CYP2C9 are important CYP enzymes which metabolise more that 60% of drugs. Induction or inhibition of the enzymatic activity and the levels of these enzymes can have significant effects on drug metabolism. Understanding the role of GR and other nuclear receptors, pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), in the mechanisms effecting CYP3A4/5 and CYP2C9 levels and activity can aid in the development of in vitro and in vivo models which have become a target for scientists in the clinic and the industry. The commonly prescribed synthetic glucocorticoid (GC) drug, dexamethasone (Dex), can induce GR, PXR and CAR and was used in this study to analyse its effects on the CYP enzymes studied. The hypothesis of this project was that changes in CYP3A4/5 and CYP2C9 gene expression affect drug metabolism and changes in gene expression of these CYP enzymes was under GR, PXR and CAR control, thus affecting the concentration and therapeutic activity of drugs metabolized by these enzymes during chronic use of GCs in conditions such as rheumatoid arthritis and asthma. This study aimed to measure mRNA, protein, ROS and enzymatic activity levels in human HepG2 hepatocytes treated with Dex for 120 h and analyze the results for various time points to produce a mathematical model. Our study has shown that changes in mRNA, protein and enzymatic activity levels of CYP3A4/5 and CYP2C9 in HepG2 cells were induced by Dex at sub-micromolar (0.1 µM) and supra-micromolar (1.5 mM) concentrations. The induction of CYP3A4/5 and CYP2C9 enzymes during 120 h treatment with Dex may be affected by the NRs studied; GR, phosphorylated GR, PXR and CAR protein levels were also shown to be induced by Dex. The efflux transporter, P-gp’s protein levels were also induced by 0.1 µM Dex, highlighting the importance of considering bioavailability of other drugs co-administered with Dex. The results of some of these laboratory experiments have been used to produce mechanistic mathematical models by MATLAB software with reference to previous studies in rats concentrating on the effects of steroids on GR. The models developed were not effective at the lower Dex concentration of 0.1 µM but were better modelled at the higher Dex concentration of 1.5 mM. The basic mechanistic models developed using HepG2 cells in this study can be utilised to design and conduct drug-drug interaction (DDI) analyses of the induction of CYP3A4/5 and CYP2C9 in other human liver cells and starting pre-clinical studies in animals to aid in drug development.

There are many benefits of predicting pharmacokinetic properties before administration to man. Recently it has become apparent that the in vivo clearance of drugs from in vitro metabolic data can be predicted with some degree of accuracy. To substantiate and extend this approach, a database of metabolic in vitro and in vivo data in rat was established for a total of 35 drugs. The database was then utilised to validate in vitro intrinsic clearance (CLint) scaling factors (1200M hepatocytes/SRW and 660mg microsomal protein/SRW). Poorly predicted drugs were then identified by applying discrimination criteria for predictive success of 50% under prediction to 100% over prediction. Of those, ethoxycoumarin (EC) and phenacetin (PNC) were chosen for closer examination with their in vivo and in vitro estimates of CLint being determined using the isolated perfused liver preparation (IPL), freshly isolated hepatocytes and hepatic microsomes. The CLints obtained for both EC and PNC from the IPL experiments under predicted the observed CLints but gave reproducible results and demonstrated the ability to reflect the trends of increasing CLint with decreasing dose and induction with beta-Naphthoflavone (betaNF). The IPL system used in the experiments was a simple one in terms of the perfusate. There were no blood cells or protein added nor was monitoring of oxygen and pH carried out. Therefore, if the IPL was to be used as a substitute for an in vivo study to determine hepatic clearance it may be necessary to use a more sophisticated system. EC depletion data from hepatocytes gave a good 84% prediction of in vivo CLint with microsomes giving a poor 38% prediction, due possibly, to end product inhibition. However, on induction with BNF, hepatocytes and microsomes both gave good predictions of 130 and 70% respectively. It was suggested that induction with BNF induces CYP isoforms which are less susceptible to end product inhibition. Predictions based upon 7-hydroxycoumarin formation data were consistent with those carried out previously and expectedly under predicted CLint. This is consistent with the existence of other pathways of metabolism. These studies demonstrated the utility of drug depletion data to predict in vivo CLint when the complete metabolic fate of a drug is unknown. The metabolism of PNC was monitored via the formation of paracetamol. When compared to the in vivo CLint, corrected for the fraction metabolised to paracetamol, hepatocytes gave a prediction of 50% with microsomes giving a prediction of 31%. End product inhibition was investigated and was shown not to be a factor in the microsomal prediction. However, recently published studies demonstrated that paracetamol could be produced from routes other than via direct O-deethylation of PNC. A further correction was made to the in vivo CLint to take into account the role of futile deacetylation which then allowed an acceptable microsomal prediction of 50% to be made. The predictions of EC and PNC CLint arising from the work carried out in this thesis were an improvement on those previously obtained by other workers. If the 50% under prediction to 100% over prediction acceptance criteria are applied, only the microsomal prediction of EC CLint would be considered as an unacceptable prediction. Use of the drug depletion approach, as demonstrated with EC, may become the method of choice to predict in vivo clearance as it overcomes metabolite identification and separation difficulties often encountered during drug metabolism studies.

Glycosaminoglycans (GAGs) are a family of complex mixture of linear polysaccharides that are present in both vertebrates and invertebrates. This polysaccharide plays important roles in physiological and pathological conditions, including cancer. In this study, GAGs were isolated from two different fish (whelks and cockles) belonging to mollusc invertebrates. The crude GAGs isolated from each shellfish demonstrated variable selective anti-cancer activities against many cancer cell lines including breast (MDANQ01 and MDA468), leukemia (MOLT-4 and K562) and ovarian (HeLa) cancer. None of the commercial GAGs exhibited any anti-cancer activities against all the cancer cells studied. Previous studies conducted on the isolation of GAGs from molluscs reported mainly its anti-coagulant and anti-inflammatory activities; thus neglecting its record of anti-cancer activity. All purified whelk fractions (A – D & F) obtained failed to show any anti-cancer activity; with the exception of fraction E, which showed equal levels of selective anti-cancer activity against breast cancer cells. Mechanism of cell death caused by the three novel GAGs on cancers cells were investigated via cell cycle analysis and apoptosis detection assay. Cell cycle analysis revealed significant perturbations in the cancer cell cycle showing cell cycle arrests at different stages. Similarly, there were significant apoptosis inductions induced by the three novel GAGs on each of the cell lines investigated. Structural elucidations of the two fish GAGs, using chemical, enzymatic, Polyacrylamide, Superose 12 size exclusion chromatography , gel filtration and SAX-HPLC methods of analysis, revealed the presence of both chondroitin sulfate (CS)/dermatan sulfate (DS)-like and HS-like GAGs. Discrepancies in the structural elucidations of the novel GAG mixtures and the commercial GAGs may be partly responsible for the anti-cancer activity of the novel polysaccharides, as changes in exogenous GAGs structural composition, especially sulfation levels or patterns, can alter its binding to growth factors, which is essential for cell proliferation.

Non-steroidal anti- inflammatory drugs (NSAIDs) have been in use for over 100 years to treat pain, exerting their analgesic effect by inhibiting prostaglandin (PG) synthesis via the COX pathway. Some of the NSAIDs have adverse side effects including ulceration of the stomach and cardiovascular events which are associated with bleeding. Search is still going on to find a safe NSAID. Two new coxib NSAIDs, namely celecoxib and etoricoxib have been developed and they exert marked beneficial effects in reducing pain in humans and other small animals with little or no side effects. No such study has been done on horses to see if they can tolerate the drug as an analgesic pain killer. This study was designed to investigate the effects of the two coxib NSAIDs, celecoxib and etoricoxib in six retired race horses to determine any adverse side effects of the drugs, the time course changes in their metabolism and elimination once administered orally in known physiological doses and the metabolites produced by each drug over time. The study employed well established clinical and biochemical techniques to measure blood-borne parameters and the metabolism of each drug. The results show that either etoricoxib or celecoxib had no adverse side effects on blood borne parameters and the stomach of the horses. Pharmacokinetic study following oral administration of 2 mg/kg b wt of either celecoxib or etoricoxib to the six race horses showed a Cmax of 1.15 ± 0.3 µg/ml, tmax, to be 4.09 ± 1.60 hr and a terminal half- life of 15.52 ± 1.99hr for celecoxib and a Cmax of 1.0± 0.09 µg/ml, tmax of 0.79 ± 0.1 hr and, terminal half- life of 11.51 ± 1.56 hr, respectively for etoricoxib. The results also show that each coxib is metabolized in the horse and both the parent drug and its metabolites are found in the urine, plasma and faeces. The results have also shown that even small traces of either drug or its metabolites can be measured in urine samples even 120 hours following oral administration. The main metabolites found in plasma, urine and faeces are hydroxyl celecoxib and carboxycelecoxib when celecoxib was administered orally to the 6 retired race horses. Similarly, hydroxymethyletoricoxib, carboxylic etoricoxib, hydroxymethyl-1-N-oxide metabolite of etoricoxib and hydroxymethyletoricoxib glucuronide were also found in plasma, urine and faeces following oral administration etoricoxib .to the animals. The results for either horse haeptocytes or camel liver show to some extend similar metabolites. In conclusion, the results show that both drugs have no adverse side effects in the horse and their metabolites are completely eliminated within 120 hours following oral administration.

Carbamazepine (CBZ), an anticonvulsant and mood-stabilising drug, is known to cause delayed type hypersensitivity reactions. These reactions occur only in a minority of patients treated with the drug, but often result in severe clinical outcomes. Although an association between CBZ-induced hypersensitivity reactions and HLA alleles has been demonstrated, the underlying mechanism(s) of toxicity are poorly understood. Cell death caused by CBZ and one of its metabolites, 9-acridinecarboxaldehyde (9-AC) was investigated. CBZ did not show cytotoxic effects in concentrations ranging from sub-therapeutic to supra-therapeutic. By contrast, 9-AC caused apoptosis in the lymphoblastoid cell line (50µM and 24 hours of exposure) and primary PBMCs (50 µM and 2 hours of exposure). PBMCs from 20 CBZ-naïve individuals showed significant inter-individual variability in the susceptibility to the cytotoxic effect of 9-AC. To further investigate the observed inter-individual variability, 331 immortalised lymphoblast cell lines of unrelated individuals from 4 populations were exposed to CBZ, CBZ-10,11 epoxide or 9-AC and cell viability was measured after 24 hour exposure. Considerable inter-individual variability in the cytotoxic response was observed for all three compounds. The genome wide association study (GWAS) revealed two genetic polymorphisms in dual oxidase 1 (DUOX1) and RP11-354|13.2 that were linked to cell toxicity at low concentrations of all three compounds. A SNP in DUOX1 was investigated further because of its biological plausibility. Genotyping of 153 patients did not show an association between this SNP and CBZ-induced hypersensitivity in Caucasians. Due to the higher than normal frequency of the DUOX1 variant in non-Caucasian patients (20%), the involvement of DUOX1 in the predisposition to CBZ-induced hypersensitivity reactions in non-Caucasians could not be excluded. To elucidate how T cell activation occurs in CBZ-induced hypersensitivity reactions, the protein binding capability of CBZ and two of its metabolites, CBZ-10,11 epoxide (CBZE) and 9-AC, to human serum albumin and glutathione S-transferase π was assessed. Only CBZE was found to bind covalently to these proteins. For 9-AC, no covalent products were observed but an indication of reversible binding was detected. Finally, newly developed genotyping methods for HLA-A*31:01 were investigated in comparison to sequence based typing. The methods were based on SSP-PCR. One of the methods showed exact accordance with the current gold standard but PCR failed to amplify the gene of interest and the control gene in considerable amount of samples (13.1%), while the second SSP-PCR typing method showed less reliability. In conclusion, the mechanisms of CBZ hypersensitivity has been investigated using a number of approaches designed to elucidate bioactivation of the drug, and how cytotoxicity links with genetic factors. The genomic approach may have the potential to identify novel biomarkers, but needs further studies with larger sample size.

The majority of drugs are prescribed on the premise that their desired and undesired effects are well characterised. However, the mechanisms underlying these effects can be elusive and are of interest to the pharmaceutical industry in terms of rational drug design. G protein-coupled receptors are a significant class of drug target that are capable of influencing multiple signalling processes, and downstream effects, simultaneously through a variety of effectors, such as G proteins or –β-arrestins. The effector activated by a given receptor is often a function of the ligand. This is termed functional selectivity and can contribute to adverse drug effects. Understanding functional selectivity in a mammalian setting is hindered by cross-talk between many competing signalling components. The Sc. cerevisiae pheromone response can be modified to isolate individual mammalian receptor- G protein interactions. Therefore, this simple organism represents an excellent tool to study functional selectivity. Further, the simplicity of this organism allows this pathway to be mathematically modelled. By applying mathematical models to mammalian GPCR signalling in yeast it is possible to extract experimentally inaccessible quantitative parameters underlying functional selectivity. This interdisciplinary approach to pharmacological mechanisms is an example of systems pharmacology. Here a systems pharmacology approach is applied to adenosine receptor signalling in yeast with a view to understanding the contribution of the ligand, receptor and G protein to functional selectivity. The first stage of this process was expression and characterisation of adenosine A1R, A2AR, A2BR and A3R subtypes in yeast. Here, the A1R and A2R subtypes were shown to be functional in yeast, but the A3R response was limited. The A1R signals through G proteins representing the inhibitory G αi family in yeast, while the A2AR and A2BR signal through both inhibitory and stimulatory G protein equivalents. Here ligand bias is quantified but further extended to describe adenosine receptor selectivity. Further, the yeast system was used to inform novel fluorescent compound development. Fluorescent ligand-binding rates would ultimately inform modelling studies. A minimal mathematical framework was developed to described A1R signalling in yeast. Ordinary differential equation models recreate dynamic cellular processes. Here an ODE model was applied to experimental time course data to predict rate constants throughout the yeast G protein cycle in the presence of the mammalian A1R. This model predicts that G protein subtype influences the ligand-receptor-G protein interactions of the A1R in yeast. Further modification of the system and fluorescent technologies may help validate these predictions.

Localised cancer therapy is a developing strategy used to overcome the systemic toxicity associated with intravenous systemic chemotherapy, which still represents the primary route of administration for the majority of current anticancer agents. Low molecular weight gelators (LMWGs) have recently gained increasing popularity as drug delivery platforms for localised cancer therapy: they are small molecules, which self-assemble into a 3D network via non-covalent interactions. Due to their inherent biocompatibility, LMWGs represent a viable alternative to the extensively explored polymer based drug delivery systems. One such drug delivery approach, based on LMWGs, involves the synthesis of gelator-based prodrugs of chemotherapeutic agents; however, to date, there remains a limited number of anticancer prodrugs that have gelation properties. Therefore, the present work aims to find new chemotherapeutic agents that display gelation properties by modification of the parent drug with known self-assembling groups. Two anticancer agents have been evaluated: the clinical antimetabolite methotrexate (MTX) and the experimental benzothiazole derivative 5F 203, both characterised by significant anticancer activity but systemic toxicity. Therefore, formulation of MTX and 5F 203 as LMWGs-based prodrugs for localised cancer therapy was considered a useful strategy to overcome the systemic toxicity of these antitumour drugs. Different synthetic approaches were explored to formulate self-assembling MTX prodrugs. The most successful one involved the synthesis of MTX derivatives bearing alkyl chains and aromatic groups at the α or both α and γ carboxylic acid terminals of MTX. Unfortunately, preliminary gelation tests performed on MTX acyl derivatives via a solvent-switch method, revealed lack of self-assembling properties. Therefore, the MTX conjugates developed were not suitable for localised drug delivery applications. Due to the absence of self-assembling properties of MTX conjugates, the potential gelation behaviour of novel derivatives of another anticancer candidate, the benzothiazole agent 5F 203, was considered, and two amide prodrugs series of 5F 203 were investigated. Amongst the compounds tested for gelation, 5F 203 succinic acid conjugate (68a) revealed formation of a hydrogel at physiological pH. Rheological measurements confirmed its LMWG nature, showing formation of a cross-linked gel network. In vitro growth inhibitory assays against breast (MCF-7) and ovarian (IGROV-1 and OVCAR-4) carcinoma cell lines showed overall activity of 5F 203 amide prodrugs in inhibiting cell proliferation. Release studies from the gel matrix of 68a revealed a release of the derivative and the active drug over 3 days, thus confirming its potential application as a depot formulation for localised delivery of 5F 203. In order to improve the rate of conversion of prodrugs into the parent amine 5F 203, compared to the previous amide series developed, acyloxyalkoxycarbonyl derivatives of 5F 203 were synthesised. Gelation tests displayed self-assembling behaviour for derivatives 76a-76c and rheological studies confirmed the LMWG nature of the new entities. The acyloxyalkoxycarbonyl prodrugs revealed in vitro potencies similar to those displayed by 5F 203, when tested against MCF-7 and IGROV-1 cell lines. The isobutyl carbamate prodrug (76a) proved to be the most potent of the series, showing hydrolysis into the active drug 5F 203, when incubated in either PBS buffer, rat or human plasma; release from the gel matrix also showed release of 5F 203 in PBS within 72 h. The outcomes from this work have therefore provided a basis for future optimisation and development of LMWG derivatives of 5F 203, as depot formulations for localised delivery of this anticancer agent.

Adverse drug reaction (ADR) reports of countries varies due to differences in the prevalence of diseases and hence the types of drugs used. ADRs are a major health and economic burden worldwide. National health authorities monitor the safety of medicines to protect consumers from the hazards of drugs. ADR databases are also maintained from where reports are regularly evaluated to detect signals of new ADRs and determine the increase of those already known. A review of paediatric and general population studies conducted on ADRs from national ADR databases was carried out. The majority of studies identified were from countries in Europe and North America, and only one study on the general population was conducted from the Ethiopian ADR database in Africa. No paediatric study was identified in Africa. Skin reactions associated with antiretroviral drugs were the most frequent ADRs in the study conducted from the Ethiopian ADR database. Anti-infective agents, mostly vaccines, were mostly associated with the ADRs in children in Europe and Latin America, and drugs used for treating attention deficit hyperactivity disorders (ADHD) were implicated with the ADRs reported for children in North America. The ADR databases of Ghana and Nigeria were analysed to evaluate the ADRs reported for children and adults. The fatalities reported and the associated drugs in the two databases were also evaluated. The ADR reporting rates for children and the general population in Ghana and Nigeria were lower than the corresponding rates observed in the review. The majority of the ADRs in Nigerian adults were reported for antiretroviral drugs, and most of those who died suffered Stevens Johnson syndrome with antimalarials as the suspect drugs. ADRs reported for Nigerian children were mainly skin reactions associated with antibiotics. Most of the reported fatalities resulted from renal failure, linked with suspected contaminated teething mixtures. Antimalarials and anthelmintics were mostly associated with the ADRs in Ghanaian adults. Most of the reported fatalities resulted from Stevens Johnson syndrome. ADRs in Ghanaian children were mostly associated with vaccines. The majority of the reported deaths resulted from unknown causes linked with antimalarials.