The application of fluorogenic and chromogenic enzymatic substrates in culture media continues to offer huge potential for accessible bacterial detection and identification. However, their clinical use poses two major disadvantages: the presence of commensal and/or coliform bacteria camouflages the detection of bacteria of interest, and false results may arise due to secretion of a similar enzyme by a different species (or strain of) that acts upon the same enzymatic substrates. In the current work, selective antimicrobial agents or suicide substrates were developed and investigated in order to prevent the growth of these interfering bacteria, thus improving the integrity of the detection and diagnosis of common clinical infections. This project has been carried out under collaboration between University of Sunderland (UK), bioMérieux (France) and Freeman Hospital, Newcastle-upon-Tyne (UK). The L-alanine analogues D/L-fosfalin and -chloro-L-alanine were synthesised, both of which target alanine racemase, and different peptide derivatives based on these moieties were prepared using the well-established IBCF/NMM coupling approach with a series of protection/deprotection methods. Two dipeptide derivatives based on para-aminobenzoic acid (PABA) were also investigated. A series of synthetic derivatives were subjected to microbiology evaluation using 20 different clinical strains of clinical bacteria at the Freeman Hospital. The stability of -Cl-L-Ala-D/L-Fos was investigated at pH 6.1 – 9.1; hydrolysis products were formed above pH 6.1. The minimum inhibitory concentration (MIC) of L-Ala-L-Ala-D/L-Fos was double the concentration of L-Ala-L-Ala-L-Fos, showing that only the diastereoisomer with L-fosfalin exhibited significant inhibitory activity against the bacteria. The introduction of -chloro-L-alanine into the phosphonopeptide derivatives enhanced the antibacterial activity; for example the growth of commensal bacterium, Escherichia coli, was inhibited at a low MIC (0.125 – 0.5 mg/L) by most X--Cl-L-Ala-D/L-Fos tripeptide analogues, except when X: sarcosine (MIC: 4 mg/L). However, no significant antibacterial activity was found in the PABA derivatives. These di- and tri-peptide fosfalin-containing derivatives, especially with the inclusion of -Cl-L-alanine, offer improved selectivity for the detection and identification of pathogenic bacteria in clinical samples by restricting the over-growth of commensal bacteria.

Many diseases are caused by pathogenic bacteria. A key example of this is sepsis, which is mostly caused by staphylococci and Gram-negative bacteria. In addition, the highly resistant ESKAPE pathogens are responsible for the majority of hospital acquired infections. In order to treat bacterial infections effectively, and to avoid promoting bacterial resistance against antibacterial drugs, the correct agents must be used, for which in turn the detection and identification of pathogenic strains is essential. This research aims to develop selective chromogenic culture media, by introducing new antibacterial agents for the improved selectivity and new chromogenic substrates for selective visualisation of certain bacterial strains. The intention of the major part of this work was to inhibit the growth of commensal bacteria in clinical samples, as they mask the growth of the infection-causing bacteria. New and known compounds were prepared for 3 evaluation as alanine racemase inhibitors. The compounds were tested on a range of clinical pathogenic and non-pathogenic bacterial strains. The molecules developed were based on the amino acid alanine and utilised bioisosteres and other replacements for the carboxylic acid moiety. Key compounds targeted included alanylmethanesulfonamide 27-L, 1-aminoethyl5-oxo-1,2,4-oxadiazole 33-L and 1-aminoethyltetrazole 32a-L. Each compound was tested initially as the alanyl-X dipeptide form. While most of the alanine bioisosteres were known structures, their novel peptide derivatives required synthetic development using both solution and solid phase techniques. The solid phase synthesis of several C-terminal 1aminoethyltetrazole peptides was successfully established by using 2-chlorotrityl chloride resin. The investigation of the antimicrobial activity of the synthesised compounds identified several clinically applicable selective inhibitors. These compounds were shown to provide differentiation between Salmonella and Escherichia coli, or enterococci and streptococci. This work also gave a useful comparison between the different alanine bioisosteres, and showed the importance of di- and oligopeptide permease systems in order to reach sufficient bacterial activity. The microbiological activity of 1- aminoethyltetrazole peptide derivatives was studied in more detail, due to their potential in clinical applications for the diagnosis of food poisoning. In other work, also directed towards the rapid and selective detection and identification of pathogenic bacteria in a clinical environment, new chromogenic substrates were prepared. Each of these compounds contained a chromogen with a phenoxazin-3-one scaffold linked to an amino acid residue. The purpose of the amino acid is to act as a unit recognised and cleaved by specific hydrolytic bacterial enzymes. Upon liberation, electronic differences between the conjugated and free forms of the chromogen resulted in the development of distinct colour changes, which provide the basis of 4 bacterial detection and identification. Synthetic methods have been developed for the efficient and economical production of this series of substrates. After preparation, these compounds were tested against a panel of clinically relevant bacteria. The aim of these substrates was to present an alternative substrate for (N-β-alanyl)-7-amino-1-pentylphenoxazine-3-one 86a, which is applied commercially in chromID® Pseudomonas aeruginosa chromogenic medium designed for the clinical detection of P.aeruginosa. The new substrates are designed to fully explore the chemical space of phenoxazinonebased chromogenic substrates, and to decrease the colour, as substrate 86a causes significant background colour in culture media. The future application of these substrates in chromogenic media resides in their potential to advance the identification of specific pathogenic bacteria and to thus facilitate the treatment of bacterial infections.

Amorphous solid dispersion (ASD) of a poorly soluble active pharmaceutical ingredient (API) in a polymeric matrix is a promising approach to increase the solubility, dissolution rate and hence bioavailability of the API. From an industrial point of view, spray-drying represents the main solvent evaporation process used for the manufacture of solid dispersions. The aim of the present PhD thesis was to evaluate the accuracy of: i) thermodynamic models (e.g. solubility parameter and Flory-Huggins theories), ii) standard screening methodologies (e.g. solvent casting and quench cooling) and iii) novel screening approaches for predicting the miscibility of binary spray-dried solid dispersions (SDSDs) so that the best performing API-polymer systems at adequate drug-loading (DL) can be selected. Two novel approaches for screening improvement, miniaturization and downscaling of regular spray-drying, were investigated and applied to API-polymer systems consisting of models drugs (Ibuprofen, Naproxen, Carbamazepine and Itraconazole) and seven polymers at four DLs. Screened samples were characterized using modulated differential scanning calorimetry (mDSC), X-ray powder diffraction (XRPD), thermo-gravimetric analysis (TGA) and scanning electron microscopy (SEM). Results obtained from miscibility and solid state characterization were compiled into principal components analysis (PCA) in order to qualitatively rank the screening approaches based upon their prediction accuracy. Non-sink dissolutions conditions with regard to the crystalline API were performed to assess the solubility enhancement and the extent of supersaturation of screened samples. The two proposed screening approaches were found to provide a greater accuracy than traditional screening methodologies to predict the miscibility of SDSDs. The limitations of theoretical models and standard screening methods tested are symptomatic of the gap existing between equilibrium solid solubility to kinetic miscibility as well as the importance of the preparation method with regard to ASD properties. Therefore, the main benefits of the novel approaches rely on their capacity to better reproduce the operating mode and process conditions of regular spray-dryer, while minimizing API needs for a production, significantly. The outcome of this work favours the downscaling approach due to its improved ability to ease the transfer from screening phases to manufacturing stage in the selection of adequate polymer and DL. In this regard, a novel three-stage decision protocol that implements spray-drying in a methodical small-scale approach for the development of ASDs during preclinical activities was developed and has successfully replaced all former practices in UCB projects.

Background Malaria remains a global health issue with the burden unevenly distributed to the disadvantage of the developing countries of the world. Nigeria, a middle-income country in sub-Saharan Africa, is one of the countries with high malaria burden in the world. As a socioeconomic issue, the high level of poverty in Nigeria is an important factor that reinforces the persistent malaria burden in the population. Poverty contributes to the malaria burden as it can affect integral aspects of malaria control like treatment seeking behaviours, access to preventive measures and treatment. Presently, there have been renewed efforts in the global malaria control resulting in reductions in the global malaria burden over the last decade. However, the development of resistance to artemisinin-based combination therapies threatens the sustainability of the present success in malaria control. The mechanism behind the development and spread of antimalarial drug resistance is a complex one with multiple factors in play. Nevertheless, antimalarial drug use behaviours remain critical drivers of drug resistance as they can affect some of the other factors. This study adopted a social epidemiological stance in exploring existing antimalarial drug use behaviours that have the potential to drive drug resistance development and spread. The study went further to investigate the role of socioeconomic factors in the adoption of the identified behaviours when treating malaria. Methods An exploratory mixed methods research design was adopted in this study. This design involved an initial systematic review of the literature to create a holistic picture of what is known about the issue under study. The systematic review informed the design of a qualitative study involving the use of interviews to explore the existing antimalarial drug use practices in the Nigerian population; and the different socioeconomic factors influencing the behaviours. The qualitative interviews informed the design of a measurement instrument and hypotheses that were tested in a survey with larger number of participants from Nigeria Findings The important malaria treatment seeking and drug use behaviours identified in this study were the practice of mixing drug for malaria treatment, presumptive treatment of malaria, sharing of malaria treatment course, and saving antimalarial drugs for future use. When symptoms are experienced, socio-economic factors, like the educational level, type of settlement, and 12 | P a g e household income level, tend to determine the treatment behaviours and therefore inform and determine the experience of malaria illness. There were statistically significant relationships between socioeconomic measures and drug use behaviours like the use of mixed drugs, stopping treatment to save drugs, sharing of antimalarial drugs, adherence to recommended dose and time of administration, presumptive treatment and use of recommended drugs for malaria treatment. These behaviours differ regarding the specific socioeconomic measures that are significantly associated with them. Discussion Physical and social environments can place constraints on an individual’s choices as well as that of a population. As shown in this study, education, income level and type of settlement, as structural factors, affect the decision on how to seek malaria treatment, what antimalarial drug to get, and how to use antimalarial drugs. Practices like mixing, stopping treatment to save drugs, and sharing of antimalarial drugs with others have the potential to encourage the development and spread of antimalarial drug resistance by exposing the parasite to sub-therapeutic doses of antimalarial drugs. Also, mixing of drugs paves the way for the sale of fake as well as expired antimalarial drugs thereby affecting malaria morbidity and illness experience. Conclusions and Recommendations In malaria campaigns, there is need to broaden the scope of antimalarial drug resistance control strategies to include strategies targeted at improving the socioeconomic status of people in malaria endemic areas. The informal health facilities were significantly associated with most of the reported resistance-promoting drug use behaviours like mixing; as such efforts to improve the way antimalarial drugs are used should target these facilities. Population-wide improvements in income level, educational level, environmental and structural conditions of the rural areas in malaria endemic settings like Nigeria, will encourage behavioural changes on how antimalarial drugs are used.

Renal transplantation is the definitive treatment for the end-stage renal failure. Despite concerted efforts to increase the number of available organs there remains a wide gap. Kidneys with small renal cell carcinoma have been used for transplantation after ex vivo resection of tumours with excellent results. Concerns regarding the behaviour of tumour under standard immunosuppression prevent this source from being popularised. We studied tumour behaviour with standard immunosuppression and immunosuppressives with anti-proliferative properties and the effect of MHC matching on tumour behaviour. Luciferase labelled Wistar rat kidney tumour cells were injected subcutaneously into Wistar or Lewis rats to mimic well and poorly matched groups. These were divided into groups receiving Cyclosporine, Sirolimus high and Sirolimus low dose and Leflunomide. Effects of matching on tumour rejection were studied by immunosuppression withdrawal in half of the animals within each group. Tumour progression was monitored with IVIS spectrum imaging system. When the immunosuppression was continued for the length of the study period with Cyclosporine immunosuppression, the tumour continued to grow in both strains. With high dose Sirolimus, the tumour was eradicated within 2 weeks in both Wistar and Lewis rats (p < 0.05). Both strains receiving low dose Sirolimus also eradicated the tumour within four weeks of treatment (p < 0.05). In Leflunomide group, 4/7 animals rejected the tumour within the 4 weeks of study period (p < 0.05). To study the effects of rejection and matching on the tumour behaviour, the immunosuppression was stopped after 2 weeks of treatment and the animals followed for another two weeks to study these effects. After treatment withdrawal, the tumour rejection was noted which was significantly stronger in poorly matched animals than in well-matched animals (p < 0.05) in cyclosporine treated animals. These results appeared to be in line with our hypothesis, that newer immunosuppressive medications with anti-neoplastic effects may be better options after transplanting kidneys after small tumour ex-vivo resection. Acute rejection showed significant ability to lead to tumour eradication, more effectively in less well-matched animals than well-matched combinations. Thus perhaps clinically, recipients of such restored kidneys should be less well matched and immunosuppressed with agents with anti-proliferative properties. These results will need to be replicated with further studies including closely monitored clinical studies before it can be popularised at a significant new source of precious organs.

Gastroretentive drug delivery systems might enhance bioavailability of some drugs formulated in sustained release dosage forms by providing a longer residence time in the stomach. The aim of this study was to develop and evaluate a swellable floatable gastroretentive drug delivery system utilizing an effervescent mechanism.

Ochratoxins are a group of nephrotoxins produced by a variety of moulds and were first described in 1965 [1]. Dietary exposure to ochratoxins represents a serious health issue and has been associated with several human and animal diseases including porcine nephropathy, Human Endemic Nephropathies and urinary tract tumours in humans. More than 20 years ago, ochratoxin A (OTA) - the most prominent member of this toxin family - was shown to be carcinogenic in rodents and since then extensive research has been performed in order to investigate whether OTA acts by a DNA reactive mode of action. Only recently, this theory has been conclusively disproven and a non-genotoxic mechanism is currently widely accepted for renal toxicity and carcinogenicity of OTA. The work presented in this thesis contributed to this field of science in various ways. First of all, new renal in vitro models were established and existing models were improved for the investigation of renal ochratoxin toxicity. Using these models, the in vitro effects of various OTA exposure scenarios were investigated and endpoints included amongst others general cytotoxicity, cell cycle analysis, protein binding and toxin transport. In all of these studies, distinct species- and sex-differences were observed which mirrored the observed effects in vivo described in literature. Furthermore, the differential toxicity of ochratoxin group members was investigated, the results of which inferred a need for specific detection of OTA and OTB. Due to the lack of such a detection tool, a new, highly specific and sensitive OTB-ELISA was successfully developed based on an OTB-specific monoclonal antibody produced and characterised in our laboratory. This tool allows screening of OTB in food and feed products, which will further improve detection and risk assessment. All of these studies contributed to the elucidation of the underlying mechanisms of ochratoxin toxicity. Therefore, this thesis can be seen as a fundamental part of a paradigm change on our understanding of how ochratoxins exert their harmful long-term effects in humans and animals, thus elucidating the ochratoxin enigma.

Despite the wide range of HPLC stationary phases available for reversed-phase high-performance liquid chromatography (RP-HPLC) and the in-depth studies using probes to highlight differences between them, there is very little in the way of stationary phases which offer selectivity that is substantially different from that offered by the very commonly used alkyl-silicas. Therefore, the primary aim of the research programme was to explore and try to exploit LC stationary phases which offered genuinely different selectivity to alkyl-silicas for typical drug applications. Chiral stationary phases (CSP) potentially had different selectivity and in this context a secondary aim was to explore aspects of the enantioselectivity of CSP as well as their chemical selectivity. Claims of orthogonal selectivity had been made for pentafluorophenyl (PFP) phases and phases exhibiting the hydrophilic interaction liquid chromatography (HILIC) mode. However, the Ultra PFP phase was found to be very similar in selectivity to ACE 5 C18 for both amitriptyline and acemetacin related compounds. The ZIC-HILIC phase was shown to behave as a reversed-phase material at high aqueous content in the mobile phase. There was some indication of selectivity orthogonal to that of ACE 5 C18 with low aqueous content in the mobile phase but this occurred at low retention and with mobile phases unsuitable for use with C18 phases in coupled (column or phase) systems. Nonetheless the work carried out shed more light on the mechanisms taking place in the HILIC mode which is currently attracting so much interest. Also it was possible to put ZIC-HILIC to good use for polar plant metabolites and other applications. Chiral stationary phases (CSP) also offered the prospect of selectivity orthogonal to that of C18 phases. Given the proliferation of such phases though and the fact that it would be useful to use CSP that gave chiral separations for a broad spectrum of compound classes as well as giving orthogonal separations between different compounds, it was decided to carry out comparative studies of CSP classes in order to identify any redundancies and to seek out CSP that were complementary to one another. The Regis Whelk-O1 CSP was shown to be much superior to other higher-generation Pirkle-concept CSP such as DACH-DNB and ULMO. Also it was shown to be complementary to the Chiralcel OD derivatised ii polysaccharide CSP and that both had something to offer alongside the widely used Chiralpak AD derivatised polysaccharide CSP. It was also found that a series of Chiralcel OD clones were virtually identical to Chiralcel OD and similarly for Chiralpak AD clones. Chiralpak IA, an immobilised version of Chiralpak AD, was not markedly less enantioselective than Chiralpak AD. Chiralcel OJ was less enantioselective than Chiralpak AD but the gap in performance was not as wide as between Whelk-O1 and the other Pirkle-concept CSP. The information gathered during these studies should prove to be of enormous value for further work in chiral LC method development screening. Before embarking on applications work utilising the stationary phase selectivity that had been found, a study was carried out on the effectiveness of the high efficiencies obtainable with short run times through ultra-performance liquid chromatography (UPLC). It was found that, for a range of pharmaceutical applications, that it was still necessary in each case to adjust selectivity before increasing speed through working at higher temperatures with faster flow rates. In the course of this work some exceptionally high speed separations for example for paroxetine and related substances, benzodiazepines and flurbiprofen and related substances, were developed. With respect to the evaluation of CSP as orthogonal phases to alkyl silicas under reversed-phase conditions, the Whelk-O1 CSP showed promise. However on closer inspection it was found that the Whelk-O1 CSP had very similar selectivity to the alkyl silica phase, ACE 5 C18, and deviation from this only occurred in instances when there was interaction with the chiral recognition site to give a separation of enantiomers. This prompted the notion that, rather than using Whelk- O1 in a coupled column system with ACE 5 C18, it could be used on its own for the separation of both trace enantiomer and all other related substances. This was shown to be possible using (S)-naproxen, laevokalim and (S)-flurbiprofen as illustrative examples. The evaluation of the enantioselectivity of CSP led to an optimised resolution (suitable for scaling up for preparative work) of the enantiomers of the former ‘legal-high’ drug, mephedrone, on Whelk-O1 under normal phase conditions. It was also shown that the infrequently used Chiralcel OJ derivatised polysaccharide iii CSP was ideal for developing an assay to determine trace amounts of (R)-nicotine in (S)-nicotine. Overall, the information obtained on stationary phase selectivity and retentivity through evaluation and application will be of great value in HPLC and UHPLC column selection and also selection of orthogonal phases for coupled column systems but, ultimately, moving forward, most value may be in aiding the design of two-dimensional LC systems for complex mixture analysis. This would particularly apply to the use of CSP with reversed-phase eluents in achiral-chiral systems.

Objective: The main goal of this study is to compare the Quality of Life (QoL) of Chronic Obstructive Pulmonary Disease (COPD) patients between three different regions: the United Arab Emirates (UAE), the United Kingdom (UK) and Morocco. Another aim of this research is to assess the predictive factors correlated to the QoL and study the extent of their involvement as well as any possible interaction between them. It is also designed to compare the QoL of control group (healthy individuals) among the same countries. Methods: A total of 1800 subjects were recruited including stable COPD patients (n=430), and a control group (n=1370) from the UK, Morocco and the UAE. St George’s Respiratory Questionnaire (SGRQ) was the instrument used to evaluate the QoL, while Mahler Dyspnoea Index was used to assess the dyspnoea. Lung functions were measured by a standardised Vitalograph spirometer, while a hand grip dynamometer was used to measure the muscle strength. Data was analysed using ANOVA Post Hoc test to compare the QoL between the centres and linear regression analysis used to assess the effect of various variables upon the QoL scores components. Extra questions were asked to the patients to study their awareness of their condition and its management, which have been tested using chi-square statistical method. Results: Our results show that differences in the QoL between the countries exist. The UK had better overall QoL than Morocco and the UAE, with no significant difference between genders. How well total SGRQ scores were predicted by BMI, dyspnoea index, total muscle grip and pulmonary function were assessed and the results indicated that age, total muscle grip, FVC% predicted, and dyspnoea index are significantly associated with the QoL. Regarding the control groups, our results indicated that there was a highly significant difference in all variables between the three countries. Conclusion: Muscle grip, age, dyspnoea and FVC% predicted are good predictors of QoL in COPD patients. Patients’ QoL deteriorates with older age and increased dyspnoea. COPD patients with poor QoL experience muscle weakness and poor lung function. There was clearly a difference between the QoL of COPD patients in the three regions and the reason behind this is mainly due to the socio-economic status and the health care system followed in each country rather than the demographic location.

Within the past fifty years, drugs from the sea have become an increasing industry for the identification and isolation of new medicinal agents. Bryozoa, sea squirts and corals are examples of many organisms that have been collected and tested for medicinal activity. Clinical testing’s of drugs such as bryostatin 1 and Et-743 have shown much success against various cancers such as kidney, prostate, and leukemia, etc. However, there are many problems affecting the economical availability of such drugs such as: 1. the potential endangerment of marine organisms due to massive quantities required for clinical use; 2. seasonal availability of the organisms and 3. numerous synthetic steps resulting in low percent yields to name a few. Detailed analyses conducted of the environment of these marine organisms resulted in the composition of chemicals that were used as an artificial property to mimic the host organisms and their environments, resulting in the cultivation of the bacteria suggestively responsible for the production of these active compounds. Other experiments conducted, involved the esterification of bryostatin 1 under various conditions, in order to show that such compounds produced, are more environmentally obtainable as opposed to being specie dependent. Computational studies binding Fe3+ to different marine natural products was also conducted in order to determine any siderophore properties that each may have. From this study cell line tests were conducted in order to determine the efficacy of a bryostatin-Fe3+ complex in comparison to bryostatin 1. Preliminary results from all the artificial media used to isolate and produce the bryostatins and Et-743 showed prominence; however, results were inconclusive due low detection values and marginal errors.