Afferent chemoreceptor activity was recorded from the peripheral cut end of the carotid sinus nerve in pentobarbitone anaesthetized cats. The effects of purines, peptides and ouabain on chemosensory activity were studied. Purines. It was found that intracarotid injections of adenosine: AMP; ADP; ATP; CoA;Me-adenosine analogues: N6-methyladenosine, 2-chloroadenosine, 3'-deoxyudenosine but not 2'-deoxyadenosine; cyclic AMP; dibutyryl cyclic AMP increased spontaneous chemoreceptor discharge. The ATP analogues, a-5- methylene ATP decreased spontaneous chemoreceptor discharge, whereas the f-y-methylene ATP caused a slight increase in discharge. Adenine and the purine nucleosides inosine and guanosine had little or no effect on the discharge. The pyrimidine nucleosides cytidine and uridine were also studied and had little or no effect on spontaneous chemoreceptor discharge. Intracarotid injection of theophylline transiently depressed spontaneous chemosensory activity and potentiated the action of adenosine. Intracarotid injection of dipyridamole increased spontaneous chemoreceptor discharge and the chemoexcitation evoked by low doses of adenosine and ATP was potentiated whereas that caused by high doses was inhibited and associated with a decrease in arterial blood pressure. Dipyridamole administered intravenously increased the chemoexcitatory actions of both low and high doses of adenosine. Responses evoked by sodium cyanide were slightly and variably modified during an adenosine infusion and those evoked by acetylcholine and dopamine were increased. It is concluded that adenosine increases chemoreceptor activity by acting on an extracellularly located receptor, which is theophylline-insensitive and probably of the 11-site type. Peptides. The opioid peptides, methionine-enkephalin, leucine-enkephalin and ;-endorphin inhibited spontaneous chemoreceptor discharge. 5-endorphin was a less potent inhibitor and the inhibition it evoked was very similar to that of morphine. Chemoinhibition induced by 5-endorphin was greatly reduced by naloxone; the inhibition associated with the enkephalins was also decreased, although not so markedly. Vasoactive intestinal polypeptide in low doses decreased spontaneous chemoreceptor activity whereas higher doses of the same substance increased chemoreceptor activity as did cholecystokinin octapeptide. Substance P was unable to overcome the chemoinhibitory effect of methionine-enkephalin. It is concluded that peptides such as methionine-enkephalin, leucine-enkephalin and vasoactive intestinal polypeptide, known to be present in the carotid body as well as others present in the brain, such as 5-endorphin and cholecystokinin octapeptide, influence carotid body chemoreceptor activity. The opioid peptides act via naloxone-sensitive receptors. Ouabain. Ouabain increased spontaneous chemoreceptor activity. During infusions the excitation was followed by a decline in discharge to frequencies near the control level. During the excitation the stimulatory action of sodium cyanide, carbon dioxide-equilibrated Locke solution and acetylcholine were potentiated. as was the chemoinhibition induced by dopamine. During the post-excitatory phase the responses evoked by these substances were reduced or abolished. It appears that ouabain has two distinct actions on the carotid body chemosensory activity: a 'sensitizing' followed by a 'desensitizing' phase. The possibility that the various substances investigated act via a common mechanism (e.g. adenylate cyclase-cyclic AMP system) is discussed.

Lipopolysaccharides (LPS) released from the outer membrane (OM) of Gram negative bacteria form aggregates which trigger an in ammatory response in humans and can result in severe septic shock. LPS molecules are heterogenic, macroamphiphilic oligosaccharides with a pro-in ammatory activity strictly related to the colloidal structures formed in solution; the morphology of the colloids formed by LPS is dependent upon the molecular conformation of the LPS monomers. The intact LPS molecule (smooth chemotype) forms elongated micelles in solution whereas truncated LPS molecules (rough chemotypes) form lamellar aggregates. Biological toxicity tests revealed that the dierent chemotypes have dierent toxic activities on cultured macrophage cells. It is believed that the micellar aggregates are responsible for the toxic eect of LPS, while the lamellar phase should not trigger an in ammatory response. The data presented in this thesis revealed that there might be a more complex connection between the toxicity of LPS and the aggregate structures. The amphiphilic cationic antimicrobial peptides (CAPs) LL37 and LFb possess putative anti-endotoxic activities and were therefore studied using small-angle neutron scattering (SANS) and cryo transmission electron microscopy (cryo-TEM) to assess any modication induced to the colloidal structure of LPS, ideally from a high toxic state to a lower toxic morphology of the aggregates. The peptides LL37 and LFb are able to induce changes to the aggregates from dierent LPS chemotypes in relation to their conformation and the LPS molecular structure. Neutron reectivity experiments on monolayer and bilayer membrane models containing dierent chemotypes of LPS were performed in the presence of the peptides; the data revealed that the two peptides have different mechanisms of interaction with membrane models containing LPS. These investigations provide new insight into the structure-activity of LPS in conditions which are more biologically-relevant than previously published data. Additionally the activity of CAPs on membrane models of the Gram negative bacteria OM was investigated to elucidate the dierent mechanisms of interaction and their possible anti-in ammatory role.

This thesis assesses the ability of a range of novel thermo-analytical techniques to determine the spatial distribution of components across the surface of pharmaceutical mini-tablets. Such information is of use in formulation development, as the surface is the point at which a solid dosage form comes into contact with the environment, and where drug stability and excipient functionality are critical. Mini-tablets provide a good model system for surface characterisation, as they have much higher surface area to volume ratios than conventionally sized tablets. Five excipients and two drugs, as powders and after compaction, were individually characterised by the following techniques: scanning electron microscopy (SEM), variable temperature infrared spectroscopy (VT-IR), differential scanning calorimetry (DSC), atomic force microscopy (AFM), micro-thermal analysis (micro-TA), nano-thermal analysis (nano-TA) and transition temperature microscopy (TTM). Compacts of mixed systems were tested using AFM, micro-TA, nano-TA and TTM, building up the complexity to 4-component systems for excipient-only mixtures and 5-component systems for drug-loaded mixtures. Micro-TA, nano-TA and TTM were able to detect each component in all of the multi-component compacts, but AFM could not differentiate between them in complex systems. The study was then repeated on realistic mini-tablet formulations, confirming these initial results. Additionally, Raman microspectroscopy was performed on the mini-tablets as a corroborative technique, this method being based on a different physical phenomenon. In conclusion, the thermal probe techniques (micro-TA, nano-TA and TTM) were shown to be sufficiently discriminating to allow the spatial mapping of components across the surface of realistic mini-tablet formulations. Hence, these techniques could be used alongside spectroscopic techniques in the analysis of complex surfaces. However, some serious issues with the automated analysis and data display functions in the TTM software were identified, which could lead to misinterpretation of the results. Potential corrective measures were suggested to alleviate these concerns and improve experimental reliability.

This thesis describes the results of experiments into the intra and inter-molecular binding of various ligands with dsDNA via the mechanism of intercalation, principally using the technique of atomic force microscopy (AFM). Since the description of the first AFM in the mid 1980’s, AFM has emerged as a sensitive and versatile analytical tool, capable both of detecting and manipulating artefacts at picometer resolutions. In these studies, AFM imaging, supported by circular dichroism, reveals unusual conformational changes in DNA that occur as a result of the binding of ligands that incorporate the acridine chromophore. These changes are distinct from those observed following the binding of other intercalators such as doxorubicin and echinomycin. Direct measurement of the length of linear DNA strands bound to acridine based ligands reveals a shortening of the DNA at very low ligand concentrations. This observation suggests that the structural changes that occur in DNA following the intercalation of the acridine chromophore are more wide ranging than previously thought and support molecular modeling studies that have proposed that the intercalated DNA duplex exhibits characteristics of both B and A form DNA. Variations in the conformational changes that occur in DNA as a result of intercalation may have implications for the application of new intercalating ligands as chemotherapeutic agents. In addition, single molecule force spectroscopy has been used to examine the capacity of bisintercalators to bind to DNA in an inter-molecular fashion. By stretching individual strands of dsDNA, acridine dimers are shown to bind to separate strands of DNA. Intermolecular binding of this kind remains an unexplored cytotoxic mechanism that may yet find an application in vivo. This observation is supported by a novel assay that utilises the controlled aggregation of gold nanoparticles. These nanoparticles, functionalised with DNA, are shown to aggregate on addition of a bisintercalator. The aggregation is fully reversible with the addition of sodium dodecylsulphate. These force spectroscopy experiments have also uncovered a previously unobserved, intermolecular binding mode of the peptide antibiotics echinomycin and TANDEM. In certain circumstances, these ligands are revealed to bind exclusively to the termini of separate DNA strands in a sequence dependent fashion. This finding may have implications for the employment of these ligands in the nanosciences, as a tool for joining short pieces of DNA or improving the efficiency of the enzymatic, blunt-end ligation of DNA.

Osmolytes affect protein stability through direct interactions with a protein, indirectly by perturbing the properties of the solvent water, and by a combination of these. In this thesis the conformational stability of the colicin E9 immunity protein (Im9) and Human serum albumin (HSA) were determined in the absence and presence of osmolytes (trehalose, sucrose, and glycerol) and their effective hydrodynamic radii measured in order to further explore the mechanism of stabilisation. Urea1/2, the midpoint of the unfolding transition, and GH2O, the free energy of unfolding, were measured in a urea-induced denaturation experiment and detected with fluorescence spectroscopy, and hydrodynamic radii (Rh) were measured with pulsed-field gradient NMR. The unfolding curves of Im9 and HSA are shifted to higher urea concentration so that Urea1/2 and GH2O increased, as the osmolyte concentration was increased indicating that Im9 and HSA are more stable in the presence of osmolytes. The Rh of Im9 and HSA increased in the presence of high concentration of trehalose and sucrose but glycerol produced a reduction. My data support the view that trehalose and sucrose act via a preferential hydration mechanism in which the water layer around the protein increases because osmolytes are excluded from the protein surface. In contrast, glycerol acts by interacting directly with the protein surface, and possibly by penetrating it, which causes the protein to become more compact as its void volume is reduced. This increase in compactness induces stabilisation. In addition, HSA formulations were studied for their stability over 6 months under various conditions using trehalose, sucrose and glycerol as stabilisers instead of acetyltryptophan and sodium octanoate, which are used commercially. However, measurements of HSA esterase-like activity and heme binding, and its aggregation state with polyacrylamide gel electrophoresis and DLS showed the osmolytes cannot stabilize the protein under high storage temperatures.

The Nrf2/Keap1 protein-protein interaction (PPI) regulates activity of the Nrf2 antioxidant and anti-inflammatory pathway. The transcription factor Nrf2 has been found to be a key mediator in the resolution of inflammation and the progression of chronic diseases. Most known inducers of the Nrf2 pathway act by covalent modification of Keap1 via electrophilic functional groups. Controlled induction of the Nrf2 pathway via specific disruption of the Nrf2/Keap1 interaction is an attractive therapeutic target. This work describes a cell penetrating, TAT-Nrf2 peptide which targets the Nrf2/Keap1 interaction in vitro. Induction of downstream genes is both sequence and dose dependent. In an established model of bacterial sepsis, the peptide reduces pro-inflammatory mediators. Investigation of both cell penetrating and Keap1 binding sequences has identified the requirements for effective Nrf2 induction in cell based assays. An in vitro purified protein, fluorescence polarisation (FP) assay was established in order to rapidly characterise these peptides. Based on the secondary structure of the Keap1 binding portion of Nrf2, further peptides were designed to constrain the conformation and mimic the full protein, while reducing overall size. Synthesis of cyclic peptides has identified the minimal sequence required for efficient binding and provides significant improvement in affinity over linear sequences. Several macrocyclisation techniques were explored in an attempt to retain biological activity, without the need for cell penetration sequences. Initially, disulfide bridge formation was used to produce peptides with affinities for Keap1 similar to the TAT-Nrf2 peptides at considerably reduced size. Subsequently, both head-to-tail cyclisation and peptide stapling were examined in order to restore potency in cell based assays. Finally, an alternative method for identification of Nrf2/Keap1 disruptors was explored. In silico docking calculations were used to identify potential novel PPI disruptors through library screening. Extracted hits were assessed using the FP assay, validating its use for high throughput screening.

Introduction: The expected outcomes from medicines are, frequently not realised due to adverse reactions, inappropriate prescribing and patient failure to take their therapy as intended. Whilst medication review provided by pharmacists is designed to address these issues evidence for the effectiveness is weak, and sometimes counterintuitive. Reasons postulated are poor study design, inappropriate intervention location and limited consultations skills demonstrated by pharmacists. This thesis is designed to develop, feasibility test and pilot a supervised medication review service for patients with type 2 diabetes (T2DM) in primary care provided by undergraduate pharmacy students as part of their undergraduate education. Method: Literature review and focus groups were undertaken to refine the intervention. Ethical approval was obtained. Medication reviews were undertaken within the medical practices and supervised by a primary care based pharmacist. Students reviewed patient’s medicines and then one-to-one medication reviews with two patients. A range of outcome measures were utilised and tested. Recruitment and attrition rates were recorded. Patient and practitioner acceptability of the intervention and education experience was obtained. Results: 5 medical practices were recruited, from which 133 patients with T2DM consented to participate with 67 randomised to the intervention group. Thirty-two students undertook 58 medication reviews with patients. Patients reported satisfaction with student-led medication reviews and information received about medicines. No improvement in patient reported medication adherence or clinical outcomes were identified. The mean change in quality of life and patients’ satisfaction with information about medicines was significantly greater in the intervention group. Pharmacy students reported increased confidence and improved communication skills. Discussion and conclusions: The feasibility and pilot study provided data which would enable delivery of a future definitive trial. The intervention was deemed acceptable by patients and demonstrated improved quality of life and satisfaction with information about medicines. Educational benefits of this study were also observed.

Poor physical stability is a limiting factor in the pharmaceutical development of APIs. Amorphous drugs are attractive due to increased dissolution; however can unpredictably revert to the thermodynamically stable crystalline form. Accurate prediction of molecular level physical stability would be advantageous. Research surrounding stability prediction is evolving; but challenging, including thermodynamic, molecular and kinetic factors. This study focussed on gaining a molecular level understanding of the structure and dynamics of amorphous dispersions using three model drugs. We aimed to develop solid-state NMR methods to provide invaluable information on molecular mobility; to ultimately use NMR for the prediction of crystallisation outcomes from stability studies, leading to quicker prediction of potential storage issues, since mobility is a key factor a�ecting stability in the amorphous state. VT solid-state NMR was used to probe di�erences in local mobility between drug and polymer; and to monitor polymorphic transitions/crystallisation in high loaded dispersions. Methodologies were veri�ed using additional physicochemical approaches. We demonstrated: • Di�erences in local mobility of drug/polymer dependent on model system and drug loading. • Miscibility detection down to 2 nm, including important temperature dependent observations. VT relaxation curves could become important visual tools for quanti�cation of drug loading and prediction of miscibility during initial development. • VT NMR was a useful tool for quick and accurate prediction of high temperature stability study outcomes • Insight into crystallisation of pharmaceuticals in formulation, demonstrating mapping complex phase transitions in high loaded dispersions • Detection of di�erent dynamic features of tolbutamide, including the identi�cation of motional processes responsible for the detection of its structural transitions VT solid-state NMR has provided a signi�cant quantity of data surrounding the mobility and stability of our systems of study. These methods provide us with a valuable characterisation `toolkit' for probing molecular mobility in solid dispersions, therefore aiding the prediction of potential long term stability issues.

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No description available.