Thesis (M.D.)--University of Hong Kong, 1978. / Also available in print.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Proefschrift--Utrecht. / Bibliography: p. 101-104.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Lung cancer accounted for 28% of all cancer related deaths in Hong Kong and has been the leading cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) is the most common lung cancer (85%) and has been linked to poor prognosis with 5-year survival rates of only 15%. Low accumulation and lack of efficient penetration of therapeutic agents in the tumor site, and severe adverse effects are the main obstacles in efficient lung cancer chemotherapy.. Triptolide (TPL), a diterpenoid triepoxide, was first isolated from the Chinese medicinal plant Tripterygium wilfordii Hook F. It had attracted extensive attention for its anti-tumor effect. However, its therapeutic potential has been limited by the poor water solubility (0.017 mg/mL) and strong toxicity with LD50 of 0.8 mg/kg. To improve the therapeutic effects and facilitate the application of TPL in lung cancer therapy, we developed different ligands-modified TPL-loaded liposomal formulations for lung cancer specific delivery.. Antibody-decorated liposomes can facilitate the precise delivery of chemotherapeutic drugs to the lung by targeting a recognition factor present on the surface of lung tumor cells. Carbonic anhydrase IX (CA IX), an enzyme overexpressed on the surface of lung cancer cells with a restricted expression in normal lungs, is used as the target for NSCLC therapy. In the present study, anti-CA IX antibody-modified TPL-loaded liposomes was developed. CA IX-directed liposomes exhibited 1.7-fold enhancement in internalization effects and 2-fold higher cytotoxicity in CA IX-positive human non-small cell lung cancer cell line A549. In vivo, CA IX-directed liposomes confined the delivery specifically to the lung and resided up to 96 h, which further showed enhanced therapeutic efficiency in orthotopic lung tumor bearing mice.. CPP33 is a tumor lineage-homing cell-penetrating peptide reported to be highly permeable into human lung cancer cell. Here, we utilized CPP33 for translocation of TPL-liposomal formulation into lung tumor cells. In vitro, CPP33-TPL-lip significantly improved apoptotic feature on A549 cells than non-modified liposomes. CPP33-lip specifically promoted the penetration ability of liposomes on A549 rather than human lung fibroblast cells (MRC-5), showing prominent cell selectivity. Furthermore, CPP33-lip showed superior penetrating ability on 3D tumor spheroids compared to non-modified liposomes.. A dual-ligand TPL-loaded liposomes (dl-TPL-lip) via conjugation of anti-CA IX antibody (targeting module) and CPP33 (trans-membrane module) was further developed to improve the therapeutic efficacy of NSCLC. The dl-TPL-lip showed superior penetrating ability and inhibiting effect on 3D tumor spheroids and significantly enhanced TPL anti-cancer efficacy following pulmonary administration in orthotopic lung cancer nude mice. The encapsulation of TPL in liposomes reduced the exposure of TPL in systemic circulation, which is demonstrated by pharmacokinetic study in rat plasma by endotracheal administration. Further anti-cancer effect study showed that dl-TPL-lip exhibited the greatest efficacy compared to TPL solution, non-modified TPL-loaded liposomes, anti-CA IX Ab or CPP33 single ligand-modified liposomes.. In summary, the findings of this study establish promising TPL delivery systems for targeted therapy of lung cancer. Current research focusing on drug delivery systems provides an insight into targeted and safe delivery of TPL in preclinical setting.

Ipratropium bromide is a short-acting, non-selective, muscarinic antagonist frequently prescribed for the treatment of Chronic Obstructive Pulmonary Disease (COPD) and as an emergency adjunct therapy for acute asthma. Within the past decade, there has been an accumulating wealth of clinical evidence which indicates that anti-muscarinic drugs, such as ipratropium, are responsible for an increased risk of stroke or, an adverse cardiovascular outcome (including increasing the risk and severity of myocardial infarction (MI)). MI remains the highest risk factor of death for COPD patients due to the systemic co-morbidities associated with COPD, which includes ischaemic heart disease (IHD). Despite the knowledge that approximately 22% of COPD patients also suffer from underlying IHD, the cardiovascular safety of muscarinic antagonists, such as ipratropium, has not been tested in a non-clinical setting of IHD or MI. In order to address this, the current project was designed to investigate, for the first time, the effects of ipratropium on the myocardium in a non-clinical setting. It was identified that under normoxic conditions, ipratropium did not have a significant effect on cardiac myocyte viability or infarction, from 3 month Sprague Dawley rats. In addition to this, following simulated ischaemia, ipratropium also did not appear to exacerbate myocardial injury. However, when ipratropium was administered in the context of simulated ischaemia followed by reperfusion, there was a significant exacerbation in myocardial injury which was characterised by increases in infarction, apoptosis, necrosis and a loss of resilience of oxidative stress. In order to characterise the mechanism by which ipratropium exerts the observed cardio-toxic effects, it was investigated whether acetylcholine (ACh) or cyclosporin A (CsA) were capable of attenuating the ipratropium induced cardiotoxicity. Both agents showed significant limitation of injury when co-administered with ipratropium indicating that ipratropium exerts its cardio-toxic effect through a mechanism which links muscarinic signalling to the mitochondrial permeability transition pore (mPTP). This supports previously published work where the protective signalling of ACh has been shown to promote the phosphorylation of pro-survival kinases, such as Akt and Erk1/2 and that this provides inhibition of the mPTP. Western blotting was employed to identify whether there was an involvement of the pro-survival kinases Akt and Erk1/2, as well as the stress induced kinase JNK. Ipratropium significantly increased levels of phospho-Akt and phospho-Erk1/2. However, JNK levels appeared to be insignificantly altered in comparison with the control groups. Both ACh and CsA were capable of limiting these increases. Further to this, an aged study was carried out, which showed that, within the aged myocardium, ipratropium is capable of eliciting further injury in comparison with the 3 month age groups. The effect of ipratropium on tolerance of oxidative stress was not significant, but, also, ACh and CsA were shown as unable to protect. Significant levels of JNK were also observed in the aged animals in comparison with the 3 month groups. In combination, the results presented here demonstrate, for the first time, that ipratropium is capable of exacerbating ischaemia/reperfusion injury in in vitro models of myocardial ischaemia/reperfusion. In addition, ACh and CsA are capable of limiting this injury, implying a role for pro-survival kinases and the mPTP in ipratropium induced myocardial injury. In the aged study, ipratropium still exacerbated injury, however, ACh and CsA appeared unable to protect, therefore promoting previous work that cellular signalling is altered in the senescent myocardium. In conclusion, further studies must be carried out in order to fully characterise the cardio-vascular safety profile of ipratropium.

No description available.

Understanding of molecular events involved in food safety and human health has become a major concern of contemporary life. Mass spectrometry (MS) is a powerful tool for characterization of complex food ingredients and biological molecules. Advances in MS-based techniques have offered new opportunities to understand the chemical changes occurring during food storage and processing as well as the molecular events perturbed by either endogenous or exogenous stimulus. In this thesis, we developed novel MS-based approaches for authentication of edible oil (i.e., edible vegetable oil, deep frying oil and gutter oil), assessment of genotoxicity of fatty acid hydroperoxides, and investigation of metabolic deregulation in pleural effusion and tissue of human lung cancer, aiming to gain a better understanding of food safety and human health. MS-based methods were developed for authentication of edible vegetable oil adulterated with used cooking oil. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that six monoglycerides could be used as the markers to discriminate the used cooking oil (e.g., deep frying oil and gutter oil) and qualified edible oil. Accumulation behavior of these six monoglycerides was detected in the repeatedly heated edible oils. Quantitation of the monoglycerides enabled authentication of commercial olive oil adulterated with a small amount of used cooking oil (approximately 1%). In addition, a matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method was developed for visual authentication of edible oil samples, including commercial vegetable oil, used cooking oil and adulterated edible oil. The method provided the capability for quantifying major chemical composition of edible oil, such as triglycerides, diglycerides and monoglycerides. The present method required minimal sample preparation and allowed screening of oil samples with high throughput (approximately 360 samples per day), providing a simple way to authenticate different types of oil samples. Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was applied to investigate the genotoxicity of two fatty acid hydroperoxides, i.e., 13-hydroperoxyoctadeca-cis-9,trans-11-dienoic acid (13-HPODE) and 13-hydroperoxy-9,11E,15Z-octadecatrienoic acid (13-HPOTE), which are important inducers of oxidative deterioration in oils and fats generated from the oxidation of linolenic acid and linoleic acid. The results demonstrated that the α,β-unsaturated aldehydes, such as 4-oxo-2-nonenal, dioxo-10-dodecenoic acid, 4-hydroperoxy-2-nonenal and 4-hydroxy-2-nonenal are the main degradation compounds of 13-HPODE and 13-HPOTE, which can covalently bound to deoxyribose-nucleosides and ribose-nucleosides to form adducts. This study provided evidences regarding the genotoxicity of fatty acid hydroperoxides at the molecular level. MS-based metabolomics methods were developed and applied for the investigation of metabolic signatures of pleural effusion and tissue of human lung cancer. A database-assisted global metabolomics method was established with utility of LC-Orbitrap MS, followed by automated mass spectral searching. The method enabled unbiased identification of 194 endogenous metabolites in pleural effusions caused by tuberculosis and malignancy. Among which, 33 differential metabolites involved in tryptophan catabolism, bile acid biosynthesis, and β-oxidation of fatty acids were found between tuberculous and malignant pleural effusions, which provided non-invasive biomarkers for diagnosis of pleural effusion samples with high sensitivity and specificity. In addition, a large-scale targeted metabolomics method was developed, which enabled reliable detection of over 400 biological metabolites, covering 92 metabolic pathways in human samples. The method was applied to characterize the metabolic profiles of non-small cell lung cancer (NSCLC) tissues. A number of distant metabolic pathways were found to be differentiated between tumor and normal tissues of lung squamous cell carcinoma and adenocarcinoma, including purine metabolism, citric acid cycle, amino acid metabolism, urea cycle, and ammonia recycling. In addition, several metabolites, such as adenosine, glutamate, glucose 1,6-bisphosphate, betaine, creatine and methionine sulfoxide were found to be associated with prognosis of NSCLC patients, which might provide potential biomarkers to monitor metabolic characteristics of NSCLC patients and treatment outcomes of the cancer.

Understanding of molecular events involved in food safety and human health has become a major concern of contemporary life. Mass spectrometry (MS) is a powerful tool for characterization of complex food ingredients and biological molecules. Advances in MS-based techniques have offered new opportunities to understand the chemical changes occurring during food storage and processing as well as the molecular events perturbed by either endogenous or exogenous stimulus. In this thesis, we developed novel MS-based approaches for authentication of edible oil (i.e., edible vegetable oil, deep frying oil and gutter oil), assessment of genotoxicity of fatty acid hydroperoxides, and investigation of metabolic deregulation in pleural effusion and tissue of human lung cancer, aiming to gain a better understanding of food safety and human health. MS-based methods were developed for authentication of edible vegetable oil adulterated with used cooking oil. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that six monoglycerides could be used as the markers to discriminate the used cooking oil (e.g., deep frying oil and gutter oil) and qualified edible oil. Accumulation behavior of these six monoglycerides was detected in the repeatedly heated edible oils. Quantitation of the monoglycerides enabled authentication of commercial olive oil adulterated with a small amount of used cooking oil (approximately 1%). In addition, a matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method was developed for visual authentication of edible oil samples, including commercial vegetable oil, used cooking oil and adulterated edible oil. The method provided the capability for quantifying major chemical composition of edible oil, such as triglycerides, diglycerides and monoglycerides. The present method required minimal sample preparation and allowed screening of oil samples with high throughput (approximately 360 samples per day), providing a simple way to authenticate different types of oil samples. Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was applied to investigate the genotoxicity of two fatty acid hydroperoxides, i.e., 13-hydroperoxyoctadeca-cis-9,trans-11-dienoic acid (13-HPODE) and 13-hydroperoxy-9,11E,15Z-octadecatrienoic acid (13-HPOTE), which are important inducers of oxidative deterioration in oils and fats generated from the oxidation of linolenic acid and linoleic acid. The results demonstrated that the α,β-unsaturated aldehydes, such as 4-oxo-2-nonenal, dioxo-10-dodecenoic acid, 4-hydroperoxy-2-nonenal and 4-hydroxy-2-nonenal are the main degradation compounds of 13-HPODE and 13-HPOTE, which can covalently bound to deoxyribose-nucleosides and ribose-nucleosides to form adducts. This study provided evidences regarding the genotoxicity of fatty acid hydroperoxides at the molecular level. MS-based metabolomics methods were developed and applied for the investigation of metabolic signatures of pleural effusion and tissue of human lung cancer. A database-assisted global metabolomics method was established with utility of LC-Orbitrap MS, followed by automated mass spectral searching. The method enabled unbiased identification of 194 endogenous metabolites in pleural effusions caused by tuberculosis and malignancy. Among which, 33 differential metabolites involved in tryptophan catabolism, bile acid biosynthesis, and β-oxidation of fatty acids were found between tuberculous and malignant pleural effusions, which provided non-invasive biomarkers for diagnosis of pleural effusion samples with high sensitivity and specificity. In addition, a large-scale targeted metabolomics method was developed, which enabled reliable detection of over 400 biological metabolites, covering 92 metabolic pathways in human samples. The method was applied to characterize the metabolic profiles of non-small cell lung cancer (NSCLC) tissues. A number of distant metabolic pathways were found to be differentiated between tumor and normal tissues of lung squamous cell carcinoma and adenocarcinoma, including purine metabolism, citric acid cycle, amino acid metabolism, urea cycle, and ammonia recycling. In addition, several metabolites, such as adenosine, glutamate, glucose 1,6-bisphosphate, betaine, creatine and methionine sulfoxide were found to be associated with prognosis of NSCLC patients, which might provide potential biomarkers to monitor metabolic characteristics of NSCLC patients and treatment outcomes of the cancer.

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