No description available.

No description available.

Aspirin is often prescribed in patients with acute coronary syndromes together with an ADP-P2Y12 inhibitor such as clopidogrel or prasugrel, an established treatment protocol called dual antiplatelet therapy. Although short lived, these drugs act irreversibly upon their targets and so are used as once-a-day treatments. The daily platelet turnover in healthy humans is approximately ten to fifteen per cent but can be considerably increased in disease conditions such as diabetes or chronic kidney disease. This leads to the daily emergence of an uninhibited subpopulation among the larger population of inhibited platelets. The aim of this thesis was the investigation of the role and contribution of this minority of uninhibited platelets in platelet aggregation and thrombus formation. Investigations found a nonlinear increase in arachidonic acid (AA)-induced aggregation in PRP containing rising proportions of uninhibited platelets mixed with aspirin-treated platelets. In contrast, stimulation of PRP containing mixed proportions of prasugrel active metabolite (PAM)-treated and uninhibited platelets by ADP showed a linear relationship between aggregatory responses and proportions of uninhibited platelets. This indicated that only uninhibited platelets would contribute to aggregate formation. However, confocal images of prelabelled platelets allowing the differentiation between inhibited and uninhibited platelets, revealed clustering of uninhibited platelets in the centre of aggregates surrounded by PAM-inhibited platelets. In contrast confocal images of uninhibited platelets combined with aspirin-treated platelets showed random, intermingled platelet distribution when stimulated by AA. Further in depth analyses by confocal microscopy and flow cytometry found the recruitment of PAM-inhibited platelets to be an active αIIbβ3-mediated process, independent of thromboxane A2 release. Whereas clustering of uninhibited platelets was not detected under flow conditions, an increase of platelet deposition with rising proportions of aspirin and/or PAM-free platelets was observed. These experiments clearly demonstrate that a general population of platelets can contain subpopulations that respond differently to overall stimulation of the population.

No description available.

Biofilm infections have been the focus of much attention and are associated with both tissue infections and medical-devices. In this study, conventional staining techniques combined with a reproducible biofilm preparation protocol were optimised for the study of single species bacterial and fungal biofilms in 96 well plates. The antibiofilm activities of the antimicrobial peptide LL-37 and its truncated mimetics KE-18 and KR-12 were evaluated and it was shown that LL-37 had significant biofilm prevention activities against Candida albicans, Staphylococcus aureus and Escherichia coli. On the other hand, KE-18, showed efficacy in biofilm prevention assays against S. aureus but not E. coli, whereas KR-12 showed no antibiofilm activities against any of the species studied. In view of the species diversity that is acknowledged to be important in biofilm related infections, a molecular method was established to facilitate the study of inter kingdom biofilm formation. Study of inter kingdom biofilms consisting of fungal and bacterial species (namely C. albicans along with one of the respiratory pathogens: Pseudomonas aeruginosa, S. aureus, or E. coli) showed that C. albicans favoured the growth of E. coli. and S. aureus, but not P. aeruginosa in dual-species biofilms. In experiments to determine the antibiofilm activities of conventional antifungal drugs it was apparent that conventional drugs were effective against single species C. albicans biofilms but that there was loss of antifungal efficacy against dual species biofilms. In assessing the antibiofilm properties of a novel family of peptide mimics, known as peptoids, antibiofilm efficacy was noted against both single and dual species biofilms. It is concluded that peptoids represent a novel class of antimicrobials that merit further study for the treatment of clinically relevant biofilms.

No description available.

Opiates have been used historically for the treatment of depression. Renewed interest in the use of opiates as antidepressants has focused on the development of kappa opioid receptor (κ-receptor) antagonists. In this thesis I have tested the hypothesis that buprenorphine/naltrexone combination has antidepressant efficacy. Buprenorphine acts as a partial mu opioid receptor (μ-receptor) agonist and a κ-receptor antagonist. By combining buprenorphine with the non-selective opioid receptor antagonist naltrexone, the idea was that the activation of μ-receptors would be reduced and the κ-antagonist properties enhanced. First, the appropriate dose of the combination that would act as a short acting κ-receptor antagonist was investigated in the mouse-tail withdrawal test. A dose of BU10119, a novel compound derived from buprenorphine, with pharmacology resembling buprenorphine/naltrexone combination was also investigated. It was established that a combination dose of buprenorphine (1 mg/kg, i.p.) with naltrexone (1 mg/kg, i.p.) functioned as a short acting κ-antagonist in the mouse-tail withdrawal test and BU10119 (1 mg/kg, i.p.) is a к-antagonist with a rapid onset and a duration of action not more than 24 hours. Furthermore, these doses of the buprenorphine/naltrexone and BU10119 were neither rewarding nor aversive in the conditioned place preference paradigm, and were without significant locomotor effects. Systemic co-administration of buprenorphine/ naltrexone (1 mg/kg, i.p.) and BU10119 in adult CD-1 male mice produced an antidepressant-like response in both the forced swim test and novelty induced hypophagia task. Behaviours in the elevated plus maze and light dark box were not significantly altered by either treatment. Moreover, pretreatment with buprenorphine/naltrexone and BU10119 blocked stress- induced analgesia in adult male CD1 mice. However, they were not capable of blocking restraint stress-induced elevation of corticosterone levels. Gene expression of k-receptor, prodynorphin and Corticotropin-Releasing Hormone Receptor 1 were not significantly altered by restraint stress or к-receptor antagonist treatment in prefrontal cortex, nucleus accumbens, hippocampus and amygdala. I propose that the combination of buprenorphine with naltrexone and BU10119, both represent a novel approaches to the treatment of depression.

Photodynamic therapy (PDT) is widely used for the treatment of skin cancer. Mechanistically, in delta-aminolevulinic acid (ALA)-mediated PDT, the addition of ALA to cells bypasses the negative feedback control of heme biosynthesis, leading to accumulation of photosensitizing concentrations of protoporphyrin IX (PPIX). Subsequent activation of cellular PPIX with an external light source (usually red light, 550-750 nm) leads to generation of reactive oxygen species (ROS), resulting in cell death. The major side effect of ALA-PDT treatment is the pain experienced by patients. Management of treatment-related pain still remains a considerable challenge in patients. Further optimization of the treatment protocol including light source, dose and duration therefore seems crucial to try and address this issue. To improve the efficiency of ALA-PDT of skin cells in the present study three approaches were used: (i) The conventional light source was changed to UVA (320-400 nm) that is absorbed more efficiently by PPIX and is 40-fold more potent in killing cultured skin cells than red light; (ii) ALA treatment was combined with the potent iron chelators, salicylaldehyde isonicotinoyl hydrazone (SIH), pyridoxal isonicotinoyl hydrazone (PIH) or desferrioxamine (DFO) to further increase the accumulation of PPIX through the depletion of iron available for ferrochelatase-mediated bioconversion of PPIX to heme; (iii) ALA treatment was combined with UVA-activatable caged iron chelators (CICs) that do not chelate iron unless activated by UVA. The CICs used were aminocinnamoyl-based SIH derivatives, ‘BY123’ and ‘BY128’. Upon activation by UVA, these CICs release the active SIH allowing for specific localised release of iron chelator in the cells. Spontaneously immortalised HaCaT cell line and Met2 cancer line (squamous cell carcinoma) were used as cell models. Cells were pre-treated (or not) for 18 h with SIH, PIH or DFO (20-100 µM), then subjected to ALA (0.5 mM) for 2 h and irradiated with low doses of UVA (5-50 kJ/m2). The quantification of intracellular PPIX was carried out by both HPLC and spectrofluorimetry after treatments of cells with ALA alone or combined with chelators. Cell death was examined 24 h after UVA exposure of ALA+/-chelators-treated cells by flow cytometry using Annexin V-propidium iodide dual staining assay. Pretreatment of HaCaT cells with ALA caused a substantial increase in the intracellular levels of PPIX which in turn sensitized the cells to very low non-cytotoxic UVA doses. Pre-treatment with DFO, PIH and SIH followed by ALA treatment further enhanced the PPIX level in HaCaT cells and caused an additional level of photosensitization to low UVA doses. Among the chelators used, SIH combined with ALA provided the most efficient increase in PPIX and cell killing following UVA irradiation, even at a lower SIH concentration of 20 µM. Among the CICs used, both UVA-activated BY123 and BY128 were as effective as SIH in increasing the level of PPIX and cell killing in ALA-treated cells following exposure to low doses of UVA. UVA-based ALA-PDT combined with SIH (or its caged-derivatives BY123 and BY128) appears therefore to be a promising modality for topical PDT. The high lipophilicity of SIH (and its caged-derivatives) which facilitates skin penetration and their potent cytotoxicity at low UVA doses should therefore allow the current modality for topical PDT to be improved, through a reduction of the time of irradiation and therefore the duration of pain experienced during the treatment. The use of SIH-based CICs will be a safer alternative to topical ALA-PDT than ‘naked’ SIH, as application of these pro-chelators will substantially decrease the exposure of the surrounding normal skin tissue to strong iron chelators and their toxic side effects.

Biopharmaceuticals represent a variety of biological products for diagnostic and therapeutic purposes, of which developments are driven by the recent advances in biotechnology. However, oral administration of biopharmaceuticals is challenging due to the low absorption of macromolecules and the potential degradation that occurs in the harsh environment of the gastrointestinal tract. Various strategies have been explored to improve the bioavailability of these therapeutic agents, and some strategies have the potential to protect these fragile proteins from the harsh environment. The low permeability of these macromolecules across the intestinal epithelium remains the main challenge. This work focuses on providing a strategy to increase the intestinal epithelium permeability of biopharmaceuticals. In nature, bacterial toxins, such as Pseudomonas aeruginosa exotoxin A (PE), are able to travel across the intestinal epithelium and target the immune cells underneath. The hypothesis of this study is that these toxins can facilitate the trans-epithelial transport of toxin-conjugated nanoparticles. Two biodegradable and mucoadhesive polymers, alginate and chitosan, were selected for the preparation of nanoparticles. Alginate-chitosan nanoparticles had a diameter of 179 ± 3 nm and a zeta potential of 17 ± 5 mV. After the conjugation with ntPE, the size and zeta potential changed to 202 ± 20 nm and -6 ± 5 mV, respectively. Transcytosis of ntPE-coupled nanoparticles was evaluated on Caco-2 cell monolayers in vitro. The transport rate was 4.36 ± 2.24 %, compared to 4.18 ± 2.05 % for nanoparticles conjugated with bovine serum albumin (BSA) and 2.83 ± 1.29 % for unmodified ones. The trans-epithelial transport of ntPE-coupled nanoparticles was then examined in the living rat small intestine. Nanoparticles coupled with ntPE were found to accumulate in the lamina propria of the rat small intestine, while no BSA-coupled nanoparticles or unmodified nanoparticles were found in the tissue sections. Finally, green fluorescent protein served as a model for biopharmaceuticals, and was loaded into alginate-chitosan nanoparticles to study the trans-epithelial delivery ability of these nano-carriers. Overall, this thesis tests the feasibility of using a bacterial toxin to facilitate the trans-epithelial transport of nano-carriers loaded with biopharmaceuticals. Results support this hypothesis and suggest these alginate-chitosan nanoparticles are worthy of continued work to develop into nano-carriers for oral administration of biopharmaceuticals.

This thesis investigated a buprenorphine (BUP) and naltrexone (NTX) combination therapy for the prevention of relapse to heroin and cocaine. Delivery of the combination by transdermal iontophoresis was also explored. In the first half of the thesis, experiments were carried out using Sprague Dawley rats; an assay of analgesia (the tail withdrawal assay) and a behavioural assay (conditioned place preference; CPP) were employed. It was found that 1.0 mg/kg of NTX (but not 0.3 mg/kg NTX) blocked the analgesic and rewarding properties of 0.3 mg/kg BUP. Interestingly, 0.3 mg/kg BUP with 3.0 mg/kg NTX, but not the same dose of NTX alone, was aversive. Using a CPP-extinction-reinstatement method, it was shown that the combination (0.3 mg/kg BUP and 1.0 mg/kg NTX), administered 10 minutes before a priming dose, prevented reinstatement of both morphine- and cocaine-seeking. In the second half of the thesis, iontophoresis (0.3 or 0.4 mA/cm2) was used to transport BUP and NTX across the skin; experiments were carried out using excised pig skin in side-bi-side glass cells. NTX transport was efficient and consistent with the standard mechanisms (electrorepulsion and electroosmosis) of iontophoresis. BUP was less efficiently delivered and accumulated in the skin; the size of the BUP skin depot increased with pH and with the concentration of BUP in the donor solution. BUP’s presence in the skin reduced convective solvent flow (indicated by a neutral marker) which had a negative impact on its flux, and also on that of NTX. The BUP/NTX combination has real potential as a relapse prevention therapy, and it is particularly exciting that it seems to be efficacious against cocaine, as treatments for cocaine dependence are desperately needed. Delivery of a therapeutically relevant amount of the combination by transdermal iontophoresis is achievable, but careful investigation is required to understand the atypical behaviour of BUP.