The formulation of hydrophobic drugs into appropriate dosage forms is challenging due to the problems associated with those drugs such as low solubility and poor dissolution. Using a liquisolid system is a promising method to improve the dissolution of hydrophobic drugs and in sustaining the release of hydrophilic drugs, in which solid drugs are dispersed in non-volatile liquid vehicles. The aim of this research was to use the liquisolid technique to enhance the dissolution rate of glibenclamide, a model hydrophobic drug, and to sustain the release of metformin-HCl, as a model hydrophilic drug. The wet granulation process was applied to liquisolid powders with the aim of overcoming issues of poor powder flowability and compressibility, especially using high viscosity liquid vehicles. This process was performed with liquisolid powders prior to compaction into tablets. Different liquisolid formulations were prepared using three liquid vehicles (polyethylene glycol400 (PEG® 400), Synperonic® PE/L44 and Cremophor® ELP), at 10 and 30 % w/w drug concentrations for glibenclamide; and 30% and 60% w/w drug concentrations for metformin-HCl. Avicel®PH102 was used as a carrier, whilst colloidal silicon dioxide was employed as a coating material to convert the wet mixtures into dry powders. Potato starch, 5% w/w, as a disintegrant was blended with the mixtures manually for 10 minutes and then 0.75% of magnesium stearate as a lubricant was added and mixed for 5 minutes. The final powder (depending on its flowability and compactability) was then compacted automatically using a single-punch tableting machine to give tablets with 4 mg for glibenclamide and 40 mg for metformin-HCl. Prepared liquisolid compacts were characterized by using British Pharmacopeia quality control tests: uniformity of weight, friability, disintegration, hardness and drug dissolution. iii It was found, for both drugs, that by application of wet granulation to liquisolid powder admixtures, the large-scale production of liquisolid compacts is feasible, which can be easily adapted to the pharmaceutical industry. In addition to enhancing the flowability and compressibility of the powders, the glibenclamide dissolution was also improved due to the enhanced binding of particles and because of the wetting effect of liquid vehicles on the hydrophobic drug, which make the drug more available for dissolution. For the sustained release preparations of liquisolid metformin-HCl, hydroxyl propyl cellulose (HPC) was used as a novel carrier in liquisolid compacts. The results showed 92% drug release after 12 hours using Cremophor®ELP (with 30% w/w drug concentration) which was the best sustained drug release formulation. Additionally, Eudragit® RL30D and Eudragit® RLPO have been used to study their effects on drug release from liquisolid formulations, examining if they can sustain or give more rapid drug release. Both types of Eudragit revealed immediate release with metformin-HCl rather than sustained drug release, with the tablets disintegrating within seconds. This suggests formulating orodispersible metformin-HCl tablets using Eudragit® RL30D as a liquid vehicle. In summary, liquisolid technology has led to promising results, not only in enhancing the drug dissolution of hydrophobic drugs, but also in sustaining and promoting the release of hydrophilic drugs.

The current study has focused on the local delivery of furazolidone to the gut with the aim of generating alternative approaches for the treatment of Helicobacter pylori (H. pylori). Furazolidone has proven antibacterial activity against H. pylori, which has a unique niche in the stomach mucus. This drug was chosen as it is not currently used for the treatment of H. pylori and thus resistance is not expected to be a problem. Chitosan micro-particles were formulated by the spray drying technique, followed by optimization of mucoadhesion and drug release profiles using glutaraldehyde crosslinking agent at two pH values (1.3 and 4.5). Results revealed that increasing glutaraldehyde decreased the mucin adsorption and at low pH drug release was increased. For liposomal formulations, the effects of furazolidone concentration, chitosan and cholesterol on encapsulation efficacy and in vitro drug release were evaluated. It was found that increasing the pH from 1.3 to 4.5 increased the mucoadhesive behaviour of chitosan coated liposomes from 42% to 60%. Also, increasing the furazolidone amount from 4mg to 5mg increased encapsulation efficiency. A combination of two antibiotics (including furazolidone) was prepared in muco-penetrative liposomal formulations; N-acetylcysteine was used for the muco- penetration effect with Pluronic F-127. These formulations were investigated for their charge effect on muco-penetration and drug encapsulation. The data showed that neutral liposomes easily diffuse through the mucus layer. Escherichia coli was selected to establish the assay protocol for Helicobacter pylori. The microdilution approach was used for assaying the furazolidone minimum inhibitory concentration (MIC), which was found to be 16 μg/ml for E. coli and 4 μg/ml for H. pylori. In time-dependent killing studies, it was possible to observe iii complete killing of the bacteria. Increasing furazolidone concentration by two fold of its MIC, reduced the time required to kill bacteria. The mucoadhesive drug formulations also increased the residence time of furazolidone in the stomach mucus from 2- 3 hours to 4-6 hours; this time period would then be appropriate for killing the bacteria in the stomach. For mucopenetration study complete killing was achieved in 2.5 hours when furazolidone with 1 % minimum inhibitory concentration of NAC which was used. Which was otherwise six hours when NAC was not added for augmentation. To conclude, delivery of furazolidone was via application of novel liposomal and spray dried formulations to either increase movement across gastric mucosa (via a muco-penetration effect) or to increase binding to the mucus (via mucoadhesive action). Hence, the various approaches used in this research have showed success (to deliver effective amounts of furazolidone locally into the stomach mucus) and the co-encapsulation of furazolidone and N-acetylcysteine is a novel approach for the delivery of antimicrobial agents to the stomach.

Trypanosoma brucei is a medically important protozoan parasite that causes African sleeping sickness in humans. Although disease treatment is possible, it is hindered by the limited availability of effective drugs and the rapid emergence of drug resistance. It is, therefore, important to identify novel drug targets for the development of new, more effective drugs. Mitochondrial carrier family (MCF) proteins transport a wide range of key metabolites across the mitochondrial inner membrane. They are important for the maintenance of key metabolic pathways in all eukaryotic cells. In particular, the MCF proteins that have been implicated in mitochondrial adenosine triphosphate (ATP) import appear to be essential for cell function and survival due to their roles in defence against oxidative stress and in the provision of the ATP required for mitochondrial electron transport and associated ATP production. Based on their important physiological roles, mitochondrial nucleotide transporters are potential novel drug targets. The main aim of this thesis was to identify and functionally characterise the putative mitochondrial nucleotide transporters in T. brucei. Sequence analysis revealed that the T. brucei genome contains 5 MCF proteins: TbMCP1, TbMCP15, TbMCP16, TbMCP20 and TbMCP23. The proteins were predicted to have roles in mitochondrial nucleotide transport based on their homology to functionally characterised MCF proteins that transport nucleotides. Of these TbMCPs, TbMCP1 has the highest sequence similarity to functionally characterised mitochondrial flavin carriers and peroxisomal ATP/adenosine monophosphate carriers in other eukaryotes, such as Flx1 in Saccharomyces cerevisiae. Sequence analysis further predicted that TbMCP15 and TbMCP16 were closely related to mitochondrial adenosine diphosphate (ADP/ATP) transporters, such as AAC2 in S. cerevisiae, whereas TbMCP20 appeared to be closely related to mitochondrial S-adenosylmethionine transporters, such as SAM5 in S. cerevisiae, and TbMCP23 appeared to be closely related to mitochondrial pyrimidine transporters, such as S. cerevisiae RIM2.

Oesophageal cancer is the eight most common cancer and the sixth leading cause of deaths worldwide. There are two major histological subtypes of oesophageal cancer, with the most predominant subtype in Europe and the USA being oesophageal adenocarcinoma (OAC). The standard of care for OAC patients with locally advanced disease is neoadjuvant therapy and surgical resection. Unfortunately, ~70% of patients do not respond to neoadjuvant therapy and non-responders gain no benefit from the aggressive treatment regimen whilst compromising their quality of life. There is an unmet clinical need for biomarkers predicative of patient's response to neoadjuvant chemoradiation therapy (neo-CRT). In a pre-treatment setting, predictive biomarkers indicative of patient response could enable the stratification of patients and would ensure individual patients receive the most effective treatment. However, the greater clinical benefit for patients may come from the development of new or combination therapeutics for OAC. Novel chemosensitising and radiosensitising therapeutics could be administered with neo-CRT to enhance tumour sensitivity, improve CRT efficacy and increase survival rates for OAC patients. MicroRNAs (miRNA/miRs) are short non-coding RNA that function to regulate gene expression at the post-transcriptional level. A single miRNA can potentially target hundreds or thousands of mRNA and subsequently alter the expression of multiple genes and proteins. As essential regulators of gene expression, miRNA are involved in all cellular processes and are dysregulated in cancer and other diseases. Furthermore, miRNA have been identified as predictors and modifiers of tumour sensitivity to chemotherapy and radiotherapy in numerous cancer types. Playing a causal role in disease development and progression, miRNA are promising biomarkers and therapeutic targets. In this study miRNA were investigated as biomarkers of OAC patient response to neo-CRT and as potential therapeutic targets through which to enhance tumour sensitivity to neo-CRT. In pre-treatment OAC biopsies, 67 miRNA that were differentially expressed between responders and non-responders to neo-CRT were identified. MiR-330-5p and miR-187 were downregulated in the pre-treatment biopsy samples of the neo-CRT non-responders. The functional roles of miR-330-5p and miR-187 were investigated as modulators of tumour sensitivity to CRT. In vitro the silencing of miR-330-5p enhanced, albeit subtly, cellular resistance to radiation. Furthermore, silencing miR-330-5p altered the expression of extracellular proteases and protease inhibitors, including MMP1. In vivo a pilot study indicated miR-330-5p silencing enhanced tumour growth and may alter tumour sensitivity to cisplatin. In vitro miR-187 overexpression enhanced cellular sensitivity to radiotherapy and cisplatin, implying that the downregulated miR-187 expression in the non-responders may confer resistance to CRT. Furthermore, miR-187 induced apoptosis in vitro and induction of apoptosis is a potential mechanism by which miR-187 enhances radiosensitivity. MiR-187 altered the expression of genes encoding extracellular proteins, including C3 and other immune related genes. Both miR-330-5p and miR-187 are potential regulators of the secretome, thus emphasising the role of miRNA as modulators of the tumour microenvironment. This study has identified miR-330-5p and miR-187 as potential therapeutic targets that could augment OAC tumour sensitivity to neo-CRT.

Cancer accounts for the highest amounts of disease related premature deaths worldwide with 8.1 million of deaths being associated with malignancy. In the cancer microenvironment, particularly in solid tumours, hypoxia plays a significant role in progression and metastasis by altering signal transduction and gene regulation which leads to aggressive phenotypes, poor prognosis and lower survival rates. Hypoxia-Inducible Factor (HIF) driven gene regulation is well established and believed to promote survival of tumour cells under the hypoxic microenvironment. Accumulating evidence also suggests that Specificity protein (Sp) family transcription factors might also play a role in the hypoxic microenvironment by regulating transcription of key hypoxia responsive genes such as VEGFA in either a HIF dependent or independent manner. In normal cells, Sp transcription factors are ubiquitously expressed and known to regulate numerous genes involved in vital cellular pathways such as cell cycle, apoptosis and angiogenesis. In tumour environments, deregulated Sp protein levels have been demonstrated and shown to correlate with poor prognosis and treatment response. However, the exact role of Sp transcription factors in hypoxic microenvironment is not fully understood. This study aimed to identify the effect of severe (chronic) hypoxia on Sp transcription factors and hypoxia-responsive gene regulation using breast cancer as a cell model. Initial studies measured the expression levels and binding activity of Sp transcription factors. Subsequently, an integrative genomic analysis was performed to identify Sp driven hypoxia-responsive genes in breast cancer cells. The study was further extended to analyse the binding kinetics of Sp protein inhibitors using surface plasmon resonance spectroscopy. Finally, transcriptional changes of hypoxia responsive genes were examined after addition of Sp inhibitors or knockdown of Sp1 level under the hypoxic environment. Expression analysis of Sp family members (Sp1-4) showed that the transcript levels of Sp genes were unaffected due to chronic hypoxic exposure whilst Sp protein levels were induced in all three cell lines. However, expression patterns were dependent on tissue type and severity of hypoxia. Twenty genes were identified as potential Sp driven hypoxia responsive genes which are consist of GC-rich putative Sp binding sites in their promoters. Gene expression analysis validated the hypoxic induction of these genes and their dependency on Sp protein-mediated transcription. Affinity studies of Sp protein inhibitors prove binding of antibiotic derivatives, Mithramycin A and Chromomycin A to GC-rich regions with different binding affinities and kinetics. Different Equilibrium constants (Kd) of Mithramycin A and Chromomycin A were identified which varied according to the promoter sites (10−3 to 10−6 M range). Furthermore, novel data also confirm the Mithramycin-DNA interaction is independent of cation Mg2+ which has been considered obligatory for DNA interaction. Interestingly, nordihydroguaiaretic acid (NDGA) derivative, Terameprocol exhibits no detectable interaction with linear DNA consisting of Sp binding sites. These results emphasise the importance of Sp proteins as regulators of hypoxia-mediated gene transcription. Sp-regulated transcription is vital in altering hypoxia-related cellular pathways and has a potential as biomarkers for solid tumours. Moreover, these results suggest the potential use of Sp antagonists to inhibit expression of key hypoxic genes in the cancer microenvironment. Results also provide solid background knowledge on pharmacokinetics of Sp inhibitors which will be useful in synthesis of new derivatives which can be used in novel therapeutic strategies for cancer and perhaps other diseases. Understanding the molecular mechanisms of Sp mediated hypoxic gene regulation can be further extended to elucidate other cellular stress and cellular adaptive mechanism.

M. Tech. Veterinary Technology. / Ensuring that biological products (specifically vaccines) are free of bacterial contamination presents a significant challenge. The European Pharmacopoeia recommends the routine use of plates, broths and a filtration system for detection of bacterial contaminants that might be present in raw materials. However, these methods could not timeously detect slow growing bacterial contaminants and a polymerase chain reaction was developed for the early detection of bacterial contamination. To eliminate false positive results the researcher compared ethidium monoazide bromide and propidium monoazide to distinguish between viable and non-viable bacterial cells, using universal primers which amplified a ±1.5 kilobase pair fragment of 16S ribosomal ribonucleic acid. The results indicated propidium monoazide more sensitive to detect low levels of viable bacteria. A comparative test between European Pharmacopoeia recommended and polymerase chain reaction methods found that the latter were the more sensitive method for contamination detection. The sequencing results of positive samples indicated the following contaminants: Stenotrophomonas spp. and Pseudomonas spp. as pre-dominant followed by Bacillus spp., Enterobacter spp., Klebsiella spp. and Propionibacterium spp. Propidium monoazide combined with polymerase chain reaction showed promise as a rapid, reproducible method for accessing cell integrity and is valuable to those who wish to employ it as an early detection method of possible contamination within a production system.

It has recently been discovered that cancer shares a link with metabolic diseases, including that of cardiovascular disease, diabetes, amongst others, where common sets of genes show similar gene expression. There is thus interest to investigate current therapies for metabolic diseases as possible anti-cancer agents. The renin-angiotensin system (RAS) regulates blood pressure and cardiovascular homeostasis through Angiotensin Converting Enzyme-1 (ACE-1) and its homolog ACE-2. RAS has also been implicated in the progression of various cancers due to the increased action of the vasoconstrictor, angiotensin II, which requires ACE-1 and specifically the Angiotensin Type 1 Receptor (AT1R) for its function. In this study, we investigated the potential association of the endogenous ACE-1 and ACE-2 enzymes in cervical cancer. Our results showed that ACE-1 and AT1R protein expression was elevated in cervical cancer cell lines compared to normal cells and that this correlated with elevated ACE-1 enzyme activity in cancer cells. Treatment with the ACE-1 inhibitors, Captopril and Lisinopril, reduced this activity. We showed that ACE-1 axis stimulation in cancer cells results in increased calcium signaling preferentially via the AT1R and this associates with cancer cell proliferation. Candesartan, an AT1R blocker significantly reduced these effects. ACE-2 expression and activity were decreased in cancer compared to normal cells. Our data shows that ACE2 activators, the natural peptide angiotensin 1-7 and small molecule Diminazene aceturate (DIZE) have anticancer effects with DIZE inducing a G2/M arrest in cancer cells. We also investigated associations between drugs targeting RAS and current chemotherapeutic agents, Cisplatin (CDDP) and Doxorubicin (DOX). Our data shows that ACE-1 axis inhibitors have an antagonistic effect on CDDP, while the ACE-2 activator DIZE associates synergistically with DOX. Taken together, these results suggest that elevated ACE- 1 expression associates with cervical cancer and that the inhibitors of ACE-1 function or activators of ACE-2 function have potential as anticancer therapies as single agents or in combination treatments with current chemotherapeutics.

Human prostatic acid phosphatase (PAcP) is a prostate epithelium- specific differentiation antigen. In prostate carcinomas, the cellular PAcP is decreased. We investigated its functional role in these cells. Several lines of evidence support the hypothesis that cellular PAcP functions as a neutral protein-tyrosine phosphatase and is involved in regulating prostate cell growth. In this study, we identify its in vivo substrate. Our results demonstrated that, in different human prostate cancer cell lines, the phosphotyrosine (Tyr(P)) level of a 185-kDa phosphoprotein (pp185) inversely correlates with the cellular activity of PAcP. On SDS-PAGE, this pp185 comigrates with the c-ErbB-2 oncoprotein. Immunodepletion experiments revealed that c-ErbB-2 protein is the major pp185 in cells. Results from subclones of LNCaP cells indicated the lower the cellular PAcP activity, the higher the Tyr(P) levels of c-ErbB-2. This inverse correlation was further observed in PAcP cDNA-transfected cells. In clone 33 LNCaP cells, L-(+)- tartrate suppresses the cellular PAcP activity and causes an elevated Tyr(P) level of c-ErbB-2 protein. Epidermal growth factor stimulates the proliferation of LNCaP cells, which concurs with a decreased cellular PAcP activity as well as an increased Tyr(P) level of c-ErbB-2. Biochemically, PAcP dephosphorylates c-ErbB-2 at pH 7.0. The results thus suggest that cellular PAcP down-regulates prostate cell growth by dephosphorylating Tyr(P) on c-ErbB-2 oncoprotein in those cells.

Activation of genes containing SRE-1 (sterol regulatory element 1) sequences is known to be under the regulation of sterols through modulation of the proteolytic maturation of SREBPs (SRE-1-binding proteins). Previous work has demonstrated SREBP-mediated transcriptional activation of genes encoding enzymes of sterol and fatty acid biosynthesis. Because synthesis of both sterols and C18 fatty acids are required for cell growth, in the absence of exogenous supplements of these lipids, we examined the hypothesis that fatty acid can also be regulatory in SREBP maturation. Our data indicate that C18 fatty acids can potentiate the biological activities of a typical, regulatory sterol: 25-hydroxycholesterol. Inhibition of C18 fatty acid synthesis in cells cultured in serum-free medium renders them resistant to killing by 25-hydroxycholesterol. Repression of expression of reporter constructs driven by promoters bearing SRE-1 element(s) by 25- hydroxycholesterol is increased by C18 fatty acid supplementation. C18 fatty acids also increase the inhibitory effect of 25-hydroxycholesterol on proteolytic maturation and nuclear localization of SREBPs. Furthermore, we also show that C18 fatty acid supplementation can enhance the inhibitory effect of 25-hydroxycholesterol on sterol and fatty acid biosynthesis. These results demonstrate that maximal down-regulation of SREBP maturation and the consequent repression of SRE-1 promoters occurs in response to both a regulatory sterol and fatty acid.

A critical step in the development of chronic inflammatory diseases is the accumulation of proinflammatory macrophages in the extracellular matrix (ECM) of peripheral tissues. The adhesion receptor integrin αDβ2 promotes the development of atherosclerosis and diabetes by supporting macrophage retention in inflamed tissue. We recently found that the end product of docosahexaenoic acid (DHA) oxidation, 2-(ω-carboxyethyl)- pyrrole (CEP), serves as a ligand forαDβ2.CEPadduct withECM is generated during inflammation-mediated lipid peroxidation. The goal of this project was to identify a specific inhibitor for αDβ2-CEP interaction that can prevent macrophage accumulation. Using a specially designed peptide library, Biacore-detected protein-protein interaction, and adhesion of integrin-transfected HEK 293 cells, we identified a sequence (called P5 peptide) that significantly and specifically inhibited αD-CEP binding. In the model of thioglycollate-induced peritoneal inflammation, the injection of cyclic P5 peptide reduced 3-fold the macrophage accumulation in WT mice but had no effect in αD-deficient mice. The tracking of adoptively transferred, fluorescently labeled WT and αD-/- monocytes in the model of peritoneal inflammation and in vitro two-dimensional and three-dimensional migration assays demonstrated thatP5peptide does not affectmonocytetransendothelial migration or macrophage efflux from the peritoneal cavity but regulates macrophage migration through the ECM. Moreover, the injection ofP5peptide intoWTmiceona high-fat diet prevents macrophage accumulation in adipose tissue in anαDβ2-dependent manner.Takentogether, these resultsdemonstratetheimportance of αDβ2-mediated macrophage adhesion for the accumulation of infiltrating macrophages in the inflamed ECM and propose P5 peptide as a potential inhibitor of atherogenesis and diabetes.