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標題: | 阿朴酚生物鹼對心肌缺血再灌流傷害之保護作用機轉與新藥開發 The Cardioprotection of Aporphine Alkaloids Against Ischemia-Reperfusion Injury and Drug Development |
作者: | Wei-Luen Chang 張瑋倫 |
指導教授: | 蘇銘嘉(Ming-Jai Su) |
關鍵字: | 缺血-再灌流傷害,心臟,阿朴酚生物鹼, Ischemia-Reperfusion Injury,Heart,Aporphine Alkaloids, |
出版年 : | 2005 |
學位: | 博士 |
摘要: | 心肌梗塞為造成死亡的主因之一,過去二十年來,由於冠狀動脈循環的重建方法逐步成熟,成為處理急性心肌梗塞的標準醫療方法,然而,缺血心肌在恢復循環時所產生所謂的再灌流傷害在此時更顯現其嚴重性,在臨床上之表現包括:心肌壞死、心律不整、心肌收縮功能不全與血管內皮或微血管失能而產生的無法再灌流現象,這些缺血-再灌流傷害的病理機制相當複雜。
在過去幾年,我們研究了對liriodenine、thaliporphine與TM-1三者阿朴酚生物鹼衍生物在不同缺血-再灌流模式下的影響與作用機制。 Liriodenine為植物Fissistiga glaucescens中分離出來的阿朴酚衍生物,過去在離體模式中顯示其抑制鈉、鉀離子管道之電生理作用為抗心律不整作用機轉,而在本論文研究的第一部分,我們評估了liriodenine在對於活體大白鼠之左冠狀動脈結紮與再灌流模式中的影響,本實驗是以動物在冠狀動脈結紮前先處理liriodenine的方式並記錄缺血或再灌流期間心律不整的發生與動物的死亡率;結果顯示:投與liriodenine在2.75x10-7g/kg的劑量下能有效預防再灌流所誘發之心律不整和死亡率,相同劑量下,也部分抑制了缺血期間心室纖維顫動之發生率與持續時間;在抗氧化活性研究中,liriodenine在1~30 uM濃度範圍內並不影響xathine oxidase的活性,同時本身對過氧自由基與DPPH並無清除能力,綜合上述結果,我們認為liriodenine能夠在心肌缺血或缺血再灌流期間抑制心律不整的發生以及減少動物的死亡率,其保護作用機轉可能與抗氧化作用或離子管道阻斷作用無關,但仍待進一步的實驗證明。 基於上述研究結果,liriodenine的心臟保護作用,一如其鈉離子與鉀離子管道阻斷作用與心臟保護作用間的關連仍待釐清;同時由於在心肌缺氧再灌流損傷時,有許多分子和細胞種類參與,因此liriodenine除了對離子管道的調控外,也可能作用於其他部分如冠狀動脈循環與血管內皮細胞來產生影響,本論文進一步針對liriodenine對於心肌與冠狀動脈內皮細胞在缺血-再灌流狀態下的保護作用與探討是否與一氧化氮途徑相關。本實驗模式採SD大白鼠以離體灌流方式,將左冠狀動脈結紮30分鐘爾後再灌流120分鐘。結果顯示:1 uM liriodenine能顯著改善冠狀動脈循環的恢復情形,同時也減少了心肌梗塞區域,此化合物能緩和內皮活性與一氧化氮釋放的減低;為模擬缺血狀態下的血管內皮細胞,進一步將人類臍靜脈內皮細胞培養在無血清條件下,結果liriodenine能隨濃度增加而經由抑制細胞在此狀態下所誘發之凋亡而增加了細胞的存活,另外,liriodenine 能減緩無血清狀態下內皮細胞或在缺血再灌流狀態下心臟的內皮一氧化氮合成酶(eNOS)的減少,同時L-NAME可抑制其對血管與心臟的保護作用,而另一個具有相似鈉、鉀管道抑制活性的化合物 quinidine,對內皮細胞與心臟細胞則無法提供保護作用。綜合以上結果liriodenine可減緩心血管在缺血再灌流狀態下的損傷而與其保護內皮一氧化氮合成酶與一氧化氮的作用有關。 Thaliporphine為另一個阿朴酚生物鹼,存在於許多藥用植物如Lauracea過去研究發現,在心肌缺血前預先處理thaliporphine能經由其抗氧化作用來減緩再灌流時的傷害。本論文更進一步評估是否在心肌缺血後一段時間後再投與thaliporphine能具有相同的保護作用,同時亦研究其其他可能的作用機轉。本實驗採活體大白鼠模式以左冠狀動脈結紮一小時後再灌流兩小時的方式,thaliporphine在再灌流前十分鐘投與,控制組投與食鹽水,另外以投與0.3 mg/kg的morphine作為實驗正對照組。實驗結果顯示:Thaliporphine 在0.05與0.5 mg/kg 的劑量下能減少心肌梗塞區域,同時在thaliporphine 0.5 mg/kg組的心臟功能恢復情形較佳,其包括有意義地改善心臟收縮速率,此化合物也可減少血中肌酐酸與心肌中MPO的活性,而thaliporphine所表現出的保護作用可以被類鴉片受體阻斷劑(naloxone or naltrexone)或粒腺體KATP 離子管道抑制劑5HD所減弱。相較之下,morphine同樣可以減少心肌梗塞區域與減少心肌中MPO的活性,但對心肌功能只有些許的恢復作用;綜合上述結果,thaliporphine可一如morphine經由活化類鴉片受體與打開粒腺體KATP 離子管道來減少再灌流期間的傷害。 最終,我們發現阿朴酚生物鹼之相關衍生物皆具明顯之心臟保護作用,並向美國申請專利(patent No. US 6313134 B1),同時在最近兩年也與美時化學公司合作進行了此心臟新藥之發展計畫,完成TM-1之臨床前藥理實驗,內容包含:1. 口服或靜脈注射前處理TM-1對心臟缺血再灌流所誘發之心律不整具有預防效果。 2. 針對臨床考量,評估TM-1對心肌缺血後再灌流前投與藥物之心臟保護效果。 3. 對TM-1在不同劑量下(包含大劑量)評估其活體心跳血壓之藥理反應,同時監測是否具急性毒性作用。 4. 完成TM-1之口服與靜脈注射之藥動學分析。 5. 對TM-1在大動物-兔子模式下進行藥理作用之確效。 綜合研究成果證實心血管之開發新藥TM-1在大白鼠與成兔兩種實驗動物模式下具有很好的心臟保護效果,在相對劑量下並不影響心跳及血壓,同時在有效劑量之10至100倍之情形下並無急性毒性作用。此計畫結果再配合完整之毒理實驗完成之後,便可以進一步進行人體臨床試驗。 Myocardial infarction is the major cause of death in the world. Over the last two decades, coronary reperfusion therapy has become an established management for acute myocardial infarction (AMI). However, restoration of blood flow to previously ischemic myocardium results in the so-called ischemia-reperfusion (I/R)-injury. The different clinical manifestations of this injury include myocardial necrosis, arrhythmia, myocardial stunning and endothelial- and microvascular dysfunction including the no-reflow phenomenon. The pathogenesis of ischemia-reperfusion injury consists of many mechanisms. In the last few years, we studied the effects of aporphine alkaloid derivatives, liriodenine, thaliporphine and TM-1, on ischemia-reperfusion models. Liriodenine is an aporphine alkaloid isolated from the plant Fissistigma glaucescens, Electrophysiological action, particularly the blockage of Na+ and K+ channels, contributes to the drug’s well-known antiarrhythmic action in the in vitro model. In the first year, we evaluated the effect of liriodenine using the coronary ligation and reperfusion models in anesthetized rats. Animals were preinfused with liriodenine before occlusion. Then the severity of ischemia and ischemia-reperfusion induced arrhythmia and mortality were investigated. We found that administration of 2.75x10-7g/kg of liriodenine is effective to prevent reperfusion induced arrhythmia and mortality. At the same dose, it also partially inhibited the incidence and duration of ventricular fibrillation induced by 30 min-myocardial ischemia. On the other hand, in the study of antioxidant activity, liriodenine (1-30uM) had no effects on xathine oxidase activity and scavenged neither superoxide anion nor diphenyl-2-picylhydrazyl (DPPH). From this study, we conclude that liriodenine is a strong cardioprotective agent, which can prevent sudden death during myocardial ischemia and ischemia-reperfusion by reducing the incidence of cardiac arrhythmia. The mechanism for its antiarrhythmic action is not related to antioxidant activity. Base on this observation, liriodenine’s cardioprotective efficacy and the relation of the channel blockages to the efficacy are needed to be clarified, as is the drug’s effect on coronary flow and endothelial function. Further study of the protective effects conveyed by liriodenine to myocardium and coronary endothelial cells under conditions of ischemia-reperfusion were performed. In the Langendorff model utilizing Sprague-Dawley rat hearts, the left main coronary artery was occluded for 30 min and reperfusion for 120 min. Liriodenine (1uM) significantly promoted the recovery of coronary flow and decreased myocardial infarction compared with vehicle-treated hearts. The drug attenuated the reduction of endothelial reactivity and NO release. To simulate the condition that occurs in the ischemic stage, human umbilical vein endothelial cells (HUVEC) were cultured in serum free conditions. Liriodenine showed concentration-dependent effects on cell viability associated with anti-apoptosis under serum-deprivation. Liriodenine prevented eNOS reduction in serum-deprived HUVEC and ischemia-reperfusion hearts. The vascular and cardioprotective effects were reversed by NG-nitro-L-arginine methyl ester. Quinidune, a Na+ and K+ channel blocker with similar activities as liriodenine failed to protect endothelial cells and myocytes. These results demonstrate that liriodenine reduces the extent of cardiovascular injuries under ischemia-reperfusion conditions mainly by preserving the eNOS and the NO production. Thaliporphine is another aporphine alkaloid found in many medicinal plants such as Lauracea. Pretreatment with thaliporphine before ischemia affords cardioprotective effects against reperfusion injury via antioxidant activity. This study was also designed to evaluate whether thaliporphine administered at a certain period after myocardial ischemia conferred the same cardioprotection and assessed its possible new mechanism. The left main coronary artery of anaesthetized rats was occluded for 1 hour and then reperfused for 2 hours. Thaliporphine was administered at 10 min before reperfusion. Controls received saline only. Morphine, a nonselective opioid receptor agonist, was used as reference compound at 0.3 mg/kg. Thaliporphine at 0.05 and 0.5 mg/kg were found to reduce the infarct size. Recovery of cardiac function was higher in thaliporphine (0.5 mg/kg) group, as assessed by a significant improvement in the rates of pressure development (+dp/dtmax). This compound also reduced plasma creatine kinase and cardiac MPO activity. These protective effects afforded by thaliporphine were diminished by the opioid receptor antagonists (naloxone or naltrexone) and by the mitochondrial KATP blocker 5HD. In comparison, morphine reduced infarct size and MPO activity in the myocardium but produced slight improvement in cardiac function after ischemia-reperfusion. These results demonstrate that reperfusion therapy with thaliporphine protect cardiac injury by mechanism via activation of opioid receptor and opening of mitochondrial KATP channels as morphine but with stronger activity. In the long run, aporphine alkaloids possess strong cardioprotective activity against ischemia and ischemia-reperfusion injury. The patent on the cardioprotection of TM-1 had been granted by the United State-patent No. US 6313134 B1. Several preclinical studies of the new cardioprotective agent-TM-1 were proceeded in last two years: 1. Pretreatment with TM-1 by either oral or intravenous administration can prevent cardiac arrhythmia and mortality in ischemia-reperfusion rats. 2. For the clinical concern, the efficacy of TM-1 was assessed when administrated at a certain period after myocardial ischemia. 3. We evaluated the hemodynamic effects of TM-1 in various dose (including high dose) and to monitor whether acute toxic effects occurred. 4. The pharmacokinetic studies of TM-1 administrated by either oral or intravenous infusion were performed. 5. The pharmacological efficacy of TM-1 was evaluated in rabbit model. Presently, we demonstrated that TM-1 reduced post-ischemic reperfusion injury both in rats and rabbits. At the effective dose, TM-1 dose not affect the blood pressure and heart rate in vivo. No experimental animal died in the study when given 10 to 100 time of effective dose of TM-1. After other toxicity tests from GLP Labs are complete, we can combine these results of TM-1 to apply for IND. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38172 |
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