An investigation of the inhibitory potential of Dronedarone on CYP2J2 mediated astemizole metabolism
Dronedarone is an anti-arrhythmic drug approved in 2009 for paroxysmal and persistent atrial fibrillation. It is less toxic than its predecessor Amiodarone as it does not cause systemic toxicity but has the same pharmacological activity. However the administration of dronedarone to permanent AF and heart failure patients leads to increased risk of stroke and cardiac death. The exact mechanism of the toxicity is currently unknown. Extrahepatic Cytochrome P450 enzymes play a dominant role in organ-specific drug metabolism and toxicity. Cytochrome P450 2J2 (CYP2J2) enzyme, a predominant enzyme found in human cardiac myocytes, metabolizes endogenous arachidonic acid (AA) into epoxyeicosatrienoic acids (EETs) which play an important role in maintaining normal cardiac physiology. Inhibition of CYP2J2 and perturbation of AA metabolic pathway could result in exacerbation of cardiac failure. This research aims to find out whether dronedarone inhibits CYP2J2 in a suitable cell model (H9C2) using astemizole as a probe substrate. Our in-house studies using recombinant CYP2J2 enzyme have shown that dronedarone potently inhibits CYP2J2. Rat myoblast cells (H9C2) will be seeded in 12-well plate and differentiated for 4 days. The cells will be then treated with different concentrations of astemizole and incubated for 24 h. The cells will then be harvested, lysed, and the cell lysate will be analyzed using liquid chromatography-mass spectrometry (LCMS). Using multi-reaction monitoring (MRM) on the LCMS, astemizole concentration as well as its CYP2J2-specific metabolite O-desmethylastemizole concentrations will be measured. The presence of O-desmethylastemizole confirms the metabolism of astemizole by CYP2J2 in H9C2 cells. By plotting a Michaelis-Menten kinetics curve, we will be able to determine the Michaelis constant (KM) and maximum rate of reaction (Vmax). H9C2 cells will be then treated with fixed concentration of astemizole while varying the dronedarone concentration. A decrease in metabolite O-desmethylastemizole conce ntration, indicates inhibition of CYP2J2 metabolism by dronedarone. Using this data, Lineweaver-Burke graph will be plotted, to determine the mode and potency of the inhibition. Our preliminary studies showed that the KM value was 2.7μM. This study will be useful in understanding if dronedarone inhibits CYP2J2 which may lead to clinically significant drug-drug interactions, one of the dangers of polypharmacy. Finally this study will shed a new light on the mechanisms for dronedarone mediated cardiac failure exacerbation.
探討抗憂鬱症藥物phenelzine對於發生在小鼠巨噬細胞中的細胞凋亡所產生的保護作用及機制
之前有研究指出，使用一些單胺氧化酶(monoamine oxidase, MAO)的抑制劑如pargyline和clorgyline，皆可以保護serum starvation所導致的細胞凋亡，表示MAO可能在細胞凋亡的路徑中扮演重要的角色。 本研究著重於一個臨床上被拿來當抗憂鬱症藥物的MAO抑制劑苯乙肼(phenelzine, PZE)對於沿著腫瘤壞死因子-α (tumor necrosis factor-α, TNF-α)途徑而產生細胞凋亡的小鼠骨髓巨噬細胞(bone marrow-derived macrophages, BMDM)所產生的保護作用。 本研究的結果顯示PZE的確可以保護循TNF-α途徑死亡的細胞，同時使活性氧化物質(reactive oxygen species, ROS)的量下降。我們推論造成此現象的原因是PZE藉由抑制MAO，使得ROS的量下降，進而保護細胞。
Novel Approach to Screening Mutations Causing Retinoblastoma, a Childhood Cancer of Retina
Retinoblastoma (RB) is a childhood retinal cancer caused by mutations in the RB1 gene. Molecular diagnosis is crucial for early detection and treatment. Current DNA diagnostic screening requires substantial amounts of tumour and blood samples. However current screening methods face the challenges of limited DNA templates from minute retinal tumours and too much blood samples drawn from young patients. In addition, the starting DNA template amount and quality are important to ensure confident detection of disease-causing mutations. As the majority of RB1 mutations are unique and distributed throughout the RB1 gene with no real hot spots, the entire gene needs to be thoroughly analysed. This investigation proposes to enrich DNA samples using a whole genome amplification (WGA) step prior to RB1 mutation screening by RB1 gene-specific PCR amplification as well as high resolution melt (HRM) analysis and sequencing. It also identifies RB1 mutations in two RB patients and explores whether WGA and saliva products can be a source of DNA templates for RB1 analysis. In addition, this study was conducted based on the hypotheses that RB1 mutations were the underlying cause of the disease in the two patients, and that the products from WGA could be used specifically for RB1 gene analysis to overcome the constraint of insufficient DNA samples. Two anonymised genomic DNA samples from two unrelated RB patients and five normal healthy DNA samples were used in this project. WGA kits were compared according to three criteria, namely amplification yield, product fragment size and whether DNA is amplifiable. Prior to and after amplification, the optical density of two normal samples was measured to determine the increase in DNA yield. The amplicons were subjected to gel electrophoresis to determine the product fragment size. Exons 6, 14 and 25 of the original and amplified samples undergone PCR, and were examined again using gel electrophoresis to ascertain that the amplicons were amplifiable. Mutation analysis using HRM was carried out with pre-existing primers for all 27 exons and the promoter of RB1. Samples from patients were analysed against 83 saliva DNAs extracted using Oragene•DNA (OG-500) Kit. REPLI-g was observed to produce higher yield and products of reliable fragment size. Single distinct bands were also seen for exons amplified using REPLI-g, indicating that REPLI-g is more accurate and suitable in the amplification of DNA. Abnormal melt profiles were obtained for exon 6 in RB477 and exon 14 in RB572 for HRM. These exons were sequenced to determine the exact mutation. Exon 6 was found to have a splice-site mutation g.607+1G>T, while a point mutation, g.1363C>T (p.Arg455X) was identified in exon 14. Both the uses of saliva as a non-invasive DNA source and the WGA approach for enriching DNA sample for application in RB1 gene analysis have never been reported for RB. Although HRM analysis has been used for other diseases, this is its first instance applied in work on RB1 gene. In short, this report offers novel and promising approaches which would contribute significantly to the molecular analysis of mutations in RB.
BA-ADA based ROS-responsive nanoparticles for selective drug delivery in cancer cells
Current medical intervention in cancer therapeutic methods has shown risks and side effects with normal tissues. This includes incomplete cancer eradication. In reference to numerous studies and literature reviews, a stimuli-responsive drug delivery system is selected as an innovative, safe and more assured treatment due to its site-specific release ability. This allows specific intervention upon the given stimulus which response to the presenting disease symptoms. Hence, we designed a ROS(Reactive Oxygen Species)-responsive BA-ADA(4-Hydroxyphenylboronic acid pinacol ester and 1-Adamantanecarboxylic acid bonded molecule) nanoparticle delivery system. In our study, ROS-responsive nanoparticle was designed and prepared based on a synthetic molecule from BA and ADA. A therapeutic payload, Doxorubicin, can be loaded into the nanoparticles and it can be selectively released within cancerous tissues whereby ROS level is over-expressed. This will enhance both therapeutic efficiency and reduce side effects. The stability and ROS-responsiveness of the particle were proven in a series of evidence-based experiments. The results showed a significant difference in cell viability during the experiments with healthy and cancerous cell samples. Further research will be required to extend the experiment in vivo.
Investigating Novel Methods to Reduce Cholesterol Levels
An increase in blood cholesterol contributes to cardiovascular diseases, the number one cause of death worldwide. Statins are currently the most effective in reducing cholesterol levels and treating patients with high cholesterol. However, these pharmaceutical agents have been shown to cause several side effects, prompting the need for a more natural solution to increasing cholesterol levels. Hence, a study was conducted to investigate the ability of lactic acid bacteria in the removal of cholesterol, explore the mechanism for the removal of cholesterol by lactic acid bacteria, and examine the effectiveness of kidney beans and sunflower seeds in inhibiting HMG-CoA reductase in the cholesterol biosynthesis pathway. Results showed that Lactobacillus plantarum was the most effective in reducing cholesterol levels and that the mechanism for cholesterol removal included both the binding to cell wall and active uptake into cells. Sunflower seeds and kidney beans were also shown to be effective in inhibiting HMG-CoA reductase, with sunflower seeds having 100% inhibition of the enzyme, similar to pravastatin, a commercial cholesterol reducing drug, and kidney beans having comparable percentage inhibition of the enzyme compared to pravastatin.