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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
11

Inter-subject variability in oral drug absorption

Rabbie, S. January 2015 (has links)
A low and highly variable bioavailability is often the main reason for the failure of the development of a drug intended for oral delivery. Focusing on absorption instead of bioavailability from oral administration enables the identification and understanding of key causes of low and erratic absorption to improve drug performance in early development. In the work carried out as part of this thesis, the in silico estimation of drug absorption (fa*fg) was carried out. The use of a population pharmacokinetic approach was proposed, as implemented in NONMEM, to estimate fa*fg and variability from phase I clinical studies (AstraZeneca database). This work enabled the identification of the rate limiting step in oral drug absorption, and allowed for comparisons of fa*fg and inter-subject variability for different drug formulations. Solubility/dissolution and permeability were investigated in vitro in terms of their variability for two model drugs – dipyridamole and furosemide. Physiological parameters such as bile salt concentrations and pH were simulated in vitro to understand their effects on the absorption process. Dipyridamole saturated solubility and dissolution are pH and bile salt dependent. However, when both dissolution and permeability were tested simultaneously, it was found that pH plays an important factor in the permeation of dipyridamole rather than bile salt concentration. This can explain to some extent the variability between individuals in the absorption of dipyridamole. Furosemide solubility experiments showed that pH, buffer capacity and, to a lesser extent, bile salt concentration affect its saturated solubility. Surprisingly, almost complete drug release was observed under all simulated conditions with a clinical dose. Similarly, the permeation of furosemide did not differ under different conditions. It was suggested that with this clinical dose, other physiological parameters contribute to variability in furosemide absorption, such as gastric emptying time. Moreover, the efficacy of three formulations (solid dispersion, Self Micro Emulsifying Drug Delivery systems and nano-particles) in increasing solubility\ dissolution in vitro and in vivo in the rat model was compared. Lack of IVIVC was observed. It was suggested that the missing link is the human absorption estimation that can be resolved by the proposed population pharmacokinetics approach presented herein.
12

Preparation of naratriptan base and evaluation of its buccal permeation characteristics

Al-Htaitawi, M. S. J. January 2015 (has links)
Naratriptan is currently delivered in tablet form for the management of migraine. Unfortunately patient compliance is poor because of gastrointestinal side effects associated with oral administration of the drug. Buccal delivery offers an attractive alternative to oral delivery as the rate of onset of drug action is faster and the incidence of gastrointestinal side effects should be reduced compared with the oral route. The aim of this project was to prepare and characterise naratriptan base and to develop and optimise a range of buccal formulations of the drug. Naratriptan was prepared from the salt form and characterised by a range of analytical techniques. In vitro permeation studies using porcine buccal mucosa were validated using caffeine as a marker molecule. Single, binary and ternary solutions, and gel formulations of naratriptan were prepared. All formulations were evaluated using the porcine buccal model. The major findings of the thesis are (i) Naratriptan base was isolated and characterised (ii) The use of porcine buccal tissue for in vitro permeation experiments was validated (iii) New dosage forms of naratriptan were successfully prepared and tested for the first time (iv) The flux values achieved for the optimal naratriptan formulation were estimated to result in therapeutic plasma concentration when applied to an area < 10 cm2.
13

Understanding the mechanisms underlying the communication between normal and tumour cells in breast cancer

Riverso, M. January 2014 (has links)
The microenvironment has been shown to play an important role in the various steps of tumour development. The main research efforts to date have focused on the tumour-stroma cross-talk, but the possibility that normal epithelial cells might also play a role in tumour progression has received little attention. The aim of this project was to study cell-cell interactions and evaluate how normal cells can influence the proliferation and apoptosis of cancer cells. Furthermore, this study aimed to delineate the signalling pathways underlying these paracrine communications and identify new potential molecular targets for therapy. To address these questions, the breast cancer cell line MCF7 and the normal mammary epithelial cells MCF10A and HMEC were used. Here, we show that non-tumorigenic mammary epithelial cells secrete factors able to enhance the proliferation of ERα+ breast cancer cells and suppress their ability to undergo apoptosis. Conditioned medium derived from MCF10A and HMEC cells (10A-CM and HMEC-CM) was capable of activating ERα in a hormone-independent manner via the PI3K/Akt/mTORC1 pathway. Finally, a cytokine antibody analysis of the conditioned media allowed the identification of MCP-1 as one of the most prevalent cytokines in the 10A-CM. We found that this cytokine recapitulates the effects of 10A-CM and HMEC-CM, as it promoted MCF7 cell proliferation. Interestingly, MCP-1 activated the ERα by phosphorylating Ser167 via PI3K/Akt/mTORC1, as demonstrated also by silencing its receptors, CCR2 and CCR4. To our knowledge, this is the first demonstration that MCP-1 is involved in the activation of ERα signalling. Together, our data indicate that normal mammary cells support the proliferation of breast cancer cells via paracrine interactions. As this communication occurs within the terminal ductal lobular units, before the cross-talk between tumour and stroma takes place, a better understanding of the role of these cells may be useful for designing drugs that will prevent the progression of the tumour at its early stages.
14

The mechanisms of metabolic regulation of the cloned equivalent of the vascular Katp/Kndp channel

Tabasum, F. January 2007 (has links)
At the molecular level, Katp is an octameric protein complex composed of an inwardly rectifying K+ channel subunit (Kir6.*) which forms the channel pore and a regulatory sulphonylurea receptor subunit (SUR). By sensing intracellular nucleotide concentrations, Katp channels couple the membrane potassium conductance of a cell to its metabolic state. To understand the molecular basis of metabolic regulation of the Katp/Kndp channel Kir6.1/SUR2B, the cell-based Rubidium-86 (86Rb+) efflux assay was employed, using HEK293 cells as the expression system. 86Rb+ efflux was activated on the addition of lOpM levcromakilim and metabolic poisoning (induced by 20mM 2-deoxyglucose and 2.5mM sodium cyanide), and inhibited by lOpM glibenclamide. The subsequent 86Rb+ distribution between intracellular and extracellular space was determined via the measurement of Cherenkov radiation, the relative amount of 86Rb+ in the cell supernatant being a direct measure of channel activity. The functionality of selected mutants was also investigated under physiological conditions by the perforated patch- clamp method, whereby currents were evoked via voltage clamp recordings over 1000ms voltage steps between -150mV and +50mV from a holding potential of-80mV. AN-Kir6.1-AC truncations were made in an attempt to understand the role of the pore- forming subunit pharmacologically in channel gating, as has previously been shown for Kir6.2AC26. Mutants for which the RXR motif was removed were surface expressed (as shown via irnmunofluorescent staining using an anti-HA-fluorescein conjugated antibody), and showed increased basal efflux in the absence of SUR2B which was reduced by inhibitors which bind to the pore-forming subunit (lmM BaCh or lOOpM PNU-37883A). However, these mutants did not display intrinsic ATP sensitivity, as for AN-K.ir6.2-AC, and for Kir6.1AC48/AN13 this was confirmed by perforated patch clamping. When Kir6.1AC48/AN13 was expressed with SUR2B, glibenclamide was able to reverse efflux induced on metabolic inhibition, but it was suspected that under this condition the sterical conformation of the channel changed which prevented Kir6.*-specific inhibitors from binding (since they were not able to reverse metabolically induced efflux). In contrast, application of either ImM BaCb or lOOuM PNU-37883A was able to reverse metabolically induced efflux for Kir6.2AC26/SUR2B. On the introduction of mutations into NBD1 or NBD2 (K708A and K1349M respectively) of SUR2B, Kir6.1/SUR2B K708A/K1349M was shown to be non-functional, whereas Kir6.2/SUR2B K708A/K1349M displayed normal pharmacology on the addition of KCOs and metabolic inhibitors. In conclusion, Kir6.1/SUR2B is sensitive to metabolic poisoning in an analogous fashion to Kir6.2/SUR2B. but the Kir6.1 pore-forming subunit does not display intrinsic metabolic sensitivity, unlike Kir6.2. Metabolic sensitivity of Kir6.1/SUR2B is determined by both nucleotide binding domains of SUR2B, and finally the pharmacology of metabolically attenuated currents is different from those activated by a Katp channel opener.
15

Hyperpolarisation-activated ion channels as a target for nitric oxide-cGMP signalling in the rat brain

Wilson, Gary William January 2008 (has links)
Most of the known physiological effects of nitric oxide (NO) in the brain are mediated by activation of specialised guanylyl cyclase-coupled receptors, leading to a rise in intracellular cGMP. Apart from protein kinase activation little is known about subsequent cGMP signal transduction. In optic nerve axons, hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels, which bind cGMP (and cAMP) directly, appear to be a target. The objective was to test this possibility directly using electrophysiological methods. Studies were initially carried out by recording extracellularly from Schaffer collateral/commissural axons in hippocampal slices, where the NO-cGMP pathway contributes to synaptic plasticity. Pharmacological manipulation of the NO-cGMP pathway failed to affect significantly axonal conduction at 0.2 - 5 Hz, a frequency range in which HCN channels were found to influence conduction reliability. Raising cAMP levels were similarly ineffective suggesting that, unlike in optic nerve, the subunit composition is likely to render the HCN channels relatively cyclic nucleotide-insensitive. Next, I investigated two neuronal types known to express the cyclic nucleotide-sensitive HCN channel subunits (HCN2 and/or HCN4), namely the principal cells of the medial nucleus of the trapezoid body and of the deep cerebellar nuclei. Using whole-cell voltage clamp, I found no reproducible evidence of regulation of HCN channel function by NO, even though exogenous cGMP was effective routinely and the neurones expressed NO-activated guanylyl cyclase, as shown by immunohistochemistry. I then carried out a series of non-invasive sharp electrode current-clamp recordings in deep cerebellar nuclear neurones. Using the characteristic voltage sag as an index of HCN channel operation, exogenous NO was found to modulate the channels reproducibly. Attempts to refine the original whole-cell recording solution to optimise preservation of the NO-cGMP pathway failed to restore NO-sensitivity. Minimising cell dialysis by using the perforated-patch variant of the whole-cell method, however, was successful. The results provide direct evidence that HCN channels are potential downstream mediators of NO-cGMP signaling in the deep cerebellar nuclei and suggest that the importance of this transduction pathway may have been previously overlooked because of unsuitable recording methods. 3.
16

Pharmaceutical applications of thermal inkjet printing

Buanz, A. B. M. January 2014 (has links)
Recent trends in the area of pharmaceutical products research and development appear to be directed more towards new drug delivery systems such as oro-dispersible films, as well as new physical forms of existing drugs such as co-crystals. Adapting technologies from other fields for developing such systems can be beneficial. Thermal inkjet printing (TIJP), a technique commonly encountered in office printers, has found applications in different areas due to its advantageous properties. The aim of this work was to utilise this technique to develop oral films for personalised dosing and to investigate its potential as a rapid and inexpensive method to prepare pharmaceutical co-crystals. Two unmodified Hewlett-Packard printers were used where the ink cartridges were modified to accommodate polymeric and aqueous drug solutions. Films were successfully prepared by printing multiple layers of hydroxyl-propyl-methylcellulose solutions onto transparency films, which disintegrated faster than those prepared by solvent casting (SC). Further, the amount deposited of a model drug (salbutamol sulphate, SS) varied linearly with the feed solution concentration. This led to the preparation of oral films using edible starch paper. Good agreement between the printed and theoretical dose was achieved with single print passes. Multiple print passes resulted in a significant loss, which was predictable and the dose variation was within the BP limits for SS tablets. Polymeric films were then used as substrates to prepare oral films of clonidine. Comparison with films prepared by solvent casting in terms of their physical stability, mechanical and drug release properties was also conducted. Films prepared by TIJP were more flexible and had better dose uniformity than films prepared by SC. Nonetheless, drug release from both films was similar. Finally, the technique was investigated to prepare pharmaceutical co-crystals. This was successfully achieved by printing solutions of co-crystal formers at specific stoichiometric ratios. In conclusion, this work demonstrates the suitability of TIJP as a method to prepare oral films extemporaneously for personalised dosing as well as pharmaceutical co-crystals.
17

Functional and structural characterisation of GABA-A receptor clustering proteins

Wesche, P. January 2014 (has links)
GABAA receptors (GABAAR) are the major inhibitory neurotransmitter receptors in the human brain. The scaffolding protein gephyrin clusters GABAAR at postsynaptic sites through a direct interaction with the GABAAR α1-3 subunits. The guanine nucleotide exchange factor collybistin facilitates translocation of gephyrin to postsynaptic sites. Symptoms of impaired GABAAR function include epilepsy and intellectual disability. The aim of my project was to further understanding of the clustering mechanisms of GABAAR using structural and molecular biology. I previously identified collybistin genes and describe here the identification of GABAAR genes in zebrafish using bioinformatics. To better understand the role of gephyrin in GABAAR clustering I purified gephyrin protein from different species for crystallisation. As partial structures for the N- and C-terminal domains are published my aim was to crystallise full-length gephyrin and the C-terminal E-domain with and without GABAAR α2 or α3 subunits. I obtained gephyrin E-domain crystal structures with a resolution of 2.3Å and 3.0Å. The improvement on the resolution and a novel space group to available E-domain structures allowed the observation of differences especially in the flexible subdomain II. Lastly, two novel collybistin mutants reported in patients with intellectual disability were analysed. Collybistin mutants R290H and R356Q showed no altered gephyrin protein interaction strength in comparison to wild-type but lost the ability to bind phosphatidylinositol 3-phosphate (PI3P) in binding assays. In addition, co-expression of collybistin mutants with gephyrin in mammalian cells showed the loss of gephyrin submembrane microcluster formation. Modelling using the published collybistin crystal structure suggested that the loss of a hydrogen bond in mutant R290H destabilise the protein structure, whereas for mutant R356Q, the substituted arginine seems to be essential for PI3P binding. In conclusion, my work provides some new insights into the role of GABAA receptor clustering proteins in health and disease.
18

Antiplasmid and antimicrobial activities of synthetic and natural products from selected medicinal plants

Mbaebie Oyedemi, B. O. January 2015 (has links)
This PhD thesis is part of ongoing project to identify plant natural products and selected synthetic compounds that possess antimicrobial properties; and are able to promote plasmid loss or interfere with bacterial conjugation. The conjugative broad host plasmids investigated include PKM101 (Inc N), TP114 (Inc I2), PUB307 (Inc P), and low- copy number plasmids: R6K (Inc X), R7K (Inc W) and R1-drd-19 (Inc F11). They represented the incompatibility plasmid groups that are currently associated with gross dissemination of antibiotic resistance in bacteria. A series of plant extracts evaluated at sub-inhibitory concentration of 100 mg/L, were shown to inhibit bacterial plasmid conjugation and their active constituents were isolated and characterised. Mallotus philippinensis yielded rottlerin and red compound, with good to moderate antibacterial activity against multidrug resistant Staphylococcus aureus strains, and had a broad range inhibition against the resistant plasmids. Investigation of extracts from the resin of Cannabis sativa L. identified tetrahydrocannabinolic acid (THCA) and cannabinolic acid (CBNA) which in addition to two synthetic cannabinoids: cannabigerol and olivetol inhibited the conjugal transfer of TP114 between E. coli strains. The antiplasmid activities of Δ9-THC, CBN, CBD, significantly reduced the transfer of amoxicillin–resistance conferring PKM 101. Methanolic extract from the dried fruits of Evodia rutaecarpa yielded evodiamine, rutaecarpine and naturally-isolated sucrose. Rutaecarpine was the most active alkaloid against NorA-expressing SA1199B and XU212 strain expressing TetK efflux mechanism. Evodiamine and sucrose had lesser antibacterial effect as well as low level of inhibition against the plasmids. Rutaecarpine and evocarpine remarkably reduced the transfer frequency of PKM 101, showing a high 2 level effect of inhibition by the compound. The bioassay-guided analysis of Capsicum annuum L. yielded capsaicin and dihydrocapsaicin (DHC) which demonstrated moderate antibacterial activities but inhibited conjugal transfer of R-plasmids actively. Capsaicin exhibited a broad range antiplasmid activity while DHC showed selective inhibition. The effect of synthetic compounds that were assessed: ferulenol, 6-gingerol and 6-shogoal were twice as effective against the transfer of PKM 101, TP114 and PUB307 compared to capsaicin, while nonivamide had no remarkable activity. With the exception of promethazine, capsaicin and dihydrocapsaicin that showed some interaction with DNA due to decreased fluorescence which suggests binding, the rest of the compounds: rottlerin, red compound, ferulenol, evocarpine, rutaecarpine, 6-gingerol, 6-shogaol and nonivamide did not bind to DNA. This may indicate other probable mechanism of antiplasmid action of the compounds. Together, some of these compounds were notable for their dual properties: robust antistaphylococcal activity and a broad host range antiplasmid effect, and are reported for the very first time. Such potentials are valuable in the discovery of next generation antimicrobial drugs.
19

Chemical and biological studies on natural and synthetic Novel Psychoactive Substances

Arunotayanun, W. January 2014 (has links)
Over the past decade, the availability of novel psychoactive substances (NPS), so called “legal highs”, has been increasing dramatically worldwide. Natural products (NP) and synthetic compounds based on NP are amongst the most commonly offered by online vendors. Most synthetic substances have recently been introduced and very little is known about their chemical and biological properties. In this project, 18 novel synthetic NPS were selected and purchased online. The full chemical characterizations using NMR, ESI-MS, HRMS, IR and UV techniques as well as in vitro neurological assays were carried out. The chemical analysis of these compounds provided a complete set of fundamental information for future analytical, forensic and synthetic work whereas the neurological results from this study made several noteworthy contributions on pharmacological and toxicological studies. One of the most valuable findings to emerge from this study is that some substances of interest including STS-135, QUPIC, 5F-QUPIC, QUCHIC, AMT, 5-MeO-DALT, 5-EAPB, 3,4-CTMP and 4-MeO-PCP, were found to exhibit moderate to high affinity interactions to at least one neurotransmitter and receptor associated with psychoactive activity suggesting that these products had a powerful potential to generate psychedelic effects in users. Moreover, some of these substances namely 5-MeO-DALT, 5-EAPB, 3,4-CTMP, 5-MAPB and MTTA, could lead to serious adverse reactions in users as they showed appreciable affinity to the 5-HT2B receptor which is known to be associated with valvular heart diseases after long term use. Another striking finding from this study was the antibacterial activities of three related isomers isolated from a commercial sample of LY-2183240, an NPS in the cannabinoid class. A 2,5-regioisomer of LY-2183240 and a novel compound, the 5,1 isomer of LY-2183240, exhibited remarkable antibacterial activities while LY-2183240 (1,5-regioisomer) was found to possess fair activity against the tested S. aureus strains, despite the fact that the structures of these three compounds were closely related. Appreciable activities were also observed for MTTA against S. aureus, as well as 5-MeO-DALT against E. coli. For natural product NPS, whilst some are well-known, without any regulation, these materials are often of dubious identity or are adulterated with other psychoactive plants or compounds, which could cause serious harm to users. Therefore, in this research, a metabolomic approach combined with multivariate data analysis was applied to examine all the metabolites present in the four top natural product NPS (Kratom, Cacti, Fly Agaric, and Hawaiian Baby Woodrose) from various UK websites. The sample preparation, data bucketing and data analysis were developed and 1H-NMR spectroscopy was selected as a tool for sample analysis due to several advantages over other methods. An interesting finding to emerge from this study was the detection of two adulterants in the kratom sample, which were identified by a metabolomic study to possess a distinct metabolite profile compared to the rest of the kratom products. These adulterants were isolated and confirmed as oxoglaucine and glaucine which have recently been classified as NPS. This clearly suggested the practical application of 1H-NMR metabolomic technique as a rapid and efficient analytical tool to produce a valuable database for the quality control of natural product NPS and for the identification of adulterants in the future.
20

Investigation of biomarkers of hepatic and renal toxicity in the Han Wistar rat

de Barros Pereira, I. M. January 2014 (has links)
The aim of this project was to identify urinary markers of hepatic and renal toxicity in the male Hanover-Wistar rat; acute and chronic injury models were developed by administration of CCl4. Nephrotoxicity was induced by administration of HCBD. In an acute dose study, CCl4-induced nephrotoxicity occurred above 2.0 mL/kg CCl4. To avoid kidney injury, 2.0 mL/kg CCl4 was chosen as the optimal dose. 1H NMR revealed many changes to the urinary metabolome following CCl4-induced liver injury including an increase in the resonances of taurine, creatine and formate and a decrease in hippurate and creatinine. Protein and gene expression markers were investigated in a HCBD-model of nephrotoxicity. Urinary α-GST, KIM-1 and albumin were the most sensitive biomarkers of proximal tubular injury. These markers could be used to detect unwanted kidney injury in future CCl4 hepatic studies. In a time course study, maximal liver injury from CCl4 was reached 24-48 hours post-dosing. Urinary metabolites followed the same trend and levels increased during the first 18-24 hours post-dosing. After 24 hours, there was a tendency for metabolites to return to control levels. A chronic model of CCl4-induced liver fibrosis was developed by dosing animals 3 times a week for 6 weeks to investigate the potential for reversibility and changes in urinary metabolites. After 6 weeks of CCl4-administration there was development of fibrous structures in the liver parenchyma followed by slight regeneration during the recovery period. Urinary metabolites that best reflected the development of fibrosis were creatinine, citrate and succinate. Taurine and hippurate may be useful for showing regenerative changes. In this project, we developed a good rat model of fibrosis which showed potential to reverse. 1H NMR analysis allowed characterisation of urinary metabolite changes in acute and chronic studies. Some of these metabolites have potential to be urinary markers for hepatic fibrosis.

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