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系統識別號 U0026-0812200914242717
論文名稱(中文) 抗黴菌唑官能基化合物對於細胞色素3A4抑制作用的機制探討
論文名稱(英文) Mechanism of CYP3A4 inhibition by antifungal azole compounds
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 96
學期 2
出版年 97
研究生(中文) 郭聖淳
研究生(英文) Sheng-chun Kuo
電子信箱 S2695105@mail.ncku.edu.tw
學號 S2695105
學位類別 碩士
語文別 中文
論文頁數 59頁
口試委員 指導教授-黃金鼎
口試委員-呂增宏
口試委員-簡偉明
中文關鍵字 抗黴菌唑官能基化合物  細胞色素3A4  類酯醇X受體 
英文關鍵字 antifungal azole compounds  CYP3A4  PXR 
學科別分類
中文摘要 藥物在人體中代謝機制主要可分為3個階段,其中作用的包括了進行氧化反應的CYP450家族、接合作用的UGT家族等酵素,以及負責將物質排出的運輸器(transporter)。過往的研究中已知Pregnane X receptor (PXR)受到外來藥物 (例如:rifampin)或是內生性物質 (例如:膽酸、脂肪酸)的活化,會與共同活化子steroid receptor coactivator-1 (SRC-1)間產生交互作用,進而調控了參與在代謝機制中的酵素以及運輸器,並且是造成臨床上藥物交互作用的原因之ㄧ。
Ketoconazole (KTZ)屬azole類抗黴菌藥物之ㄧ種,其被證實對於CYP3A4這個代謝近50%臨床上使用藥物的酵素之活性以及轉錄機制皆有抑制作用。與KTZ屬同一類抗菌藥物中包括含有imidazole官能基,例如:miconazole (MIC)以及triazole官能基,例如:Fluconazole (FLU)兩種分類的藥物。臨床上使用發現含有imidazole官能基的azole類抗黴菌藥物,其安全性較小且藥物交互作用大於含triazole官能基的azole類藥物。因此在我們的研究中,主要想探討此兩種azole類抗黴菌藥物對於CYP3A4酵素在不同層次上的抑制作用為何,是否有所差異?
我們利用HepG2細胞,以acid phosphatase (ACP) assay選擇確保細胞維持70%以上存活率的azole類藥物濃度來進行實驗。經由P450-GloTM assay及西方點墨法實驗發現,含imidazole官能基的azole類藥物在rifampin的存在下,對於CYP3A4酵素活性及蛋白表現量具明顯抑制情形,進一步以mammalian-two hybrid assay確認PXR和SRC-1間交互作用受到的抑制也會隨著藥物濃度增加而增加。然而有趣的是,azole類中含有triazole官能基的fluconazole,在臨床上用藥時發現,與其他經由CYP3A4代謝的藥物併用時,相較於含有imidazole官能基的azole類藥物以及屬同類triazole官能基的itraconazole和voriconazole,其藥物交互作用發生情形較少,因此我們以在臨床上使用所能達到之藥物血中濃度 (5~50 M)的範圍內,確認其不具有細胞毒性來進行實驗,證實其對於CYP3A4酵素活性、蛋白表現量及PXR和SRC-1間的交互作用皆不發生抑制作用。
經由我們的研究結果,發現含有imidazole官能基以及triazole官能基兩種分類的抗黴菌藥,對於代謝酵素CYP3A4在不同層次上抑制作用不盡相同,可解釋此類藥物臨床上發生藥物交互作用情形不一的原因。我們也認為藥物經由破壞PXR和共同活化子 (例如:SRC-1)間的交互作用,而使PXR所調控的酵素、運輸器表現量及活性受到改變,在藥物交互作用中是很重要的分子機制。
英文摘要 Pregnane X receptor (PXR) functions as a xenobiotic sensor which recruit transcriptional coactivators such as steroid receptor coactivator-1 (SRC-1) upon ligand binding, and therefore induces genes involves in drug metabolism and disposition, such as CYP3A4 enzyme and P-glycoprotein. Ketoconazole (KTZ), a clinically used antifungal drug, inhibits not only the activity of CYP3A4 on the enzymatic level, but also the transcription of CYP3A4. Appropriate concentration of azole compounds was selected and ensured the cell viability was about 70% for the further investigated the inhibition effect of CYP3A4 in the presence of PXR ligand. We proved the inhibition of CYP3A4 enzyme activity by all azole compounds except fluconazole. The azole compounds with imidazole functional group including miconazole, econazole and sulconazole, significantly inhibited CYP3A4 mRNA and protein level and in a dose dependent manner. But bifonazole with imidazole functional group showed slightly inhibition effect of CYP3A4 mRNA and protein level. The azole compounds dose-dependently inhibited the interaction of PXR-SRC-1 by using mammalian two-hybrid assay. Interestingly, fluconazole and voriconazole which are the azole compounds with triazole functional group, functional performs minor drug-drug interaction. It had no cytotoxicity at a clinical achievable dose (5~50 mM) and did not disrupt the interaction between PXR and SRC-1. Furthermore, only fluconazole had no inhibition effect on CYP3A4 on mRNA, protein level and enzyme activity. Itraconazole with triazole functional group destroyed CYP3A4 enzyme activity and PXR-SRC-1 interaction but did not affect the mRNA, protein level. Our present studies indicate the serious drug–drug interaction of the azole compounds with imidazole functional group may result from not only the metabolism by the hepatic CYP450 system but also the disruption of the interaction of PXR and SRC-1. Understanding the rifampin-induced PXR-mediated mechanism of CYP3A4 was suppressed by antifungal drugs with different groups may improve therapeutic drugs used to prevent adverse effect of drug-drug interaction.
論文目次 中文摘要---------------------------------------------- I
英文摘要---------------------------------------------- III
誌謝-------------------------------------------------- V
目錄-------------------------------------------------- VII
表目錄------------------------------------------------ IX
圖目錄------------------------------------------------ X
縮寫檢索表-------------------------------------------- XI
壹、緒論
一、前言---------------------------------------------- 1
二、細胞色素P450酵素家族及細胞色素P450 3A之介紹------- 1
三、CYP3A4在藥物代謝上之重要性------------------------ 2
四、類酯醇X受體(PXR)之介紹---------------------------- 4
五、藥物活化PXR誘發CYP3A4 表現的分子機轉-------------- 5
六、PXR共同活化子steroid receptor coactivator-1(SRC-1)-
------------------------------------------------------ 6
七、抗黴菌唑(azole)官能基藥物之介紹------------------- 7
八、研究目標------------------------------------------ 9
貳、實驗材料------------------------------------------ 10
参、實驗方法
一、細胞培養------------------------------------------ 16
二、實驗藥品配製-------------------------------------- 16
三、CYP3A4 酵素活性測定------------------------------- 17
四、細胞存活率測定------------------------------------ 17
五、hPXR與共同活化子SRC-1間交互作用試驗--------------- 18
六、CYP3A4 mRNA表現量分析----------------------------- 19
七、CYP3A4蛋白質表現量分析---------------------------- 21
肆、實驗結果及分析
一、抗黴菌唑(azole)官能基藥物對HepG2細胞產生之細胞毒性-
------------------------------------------------------ 23
二、抗黴菌唑(azole)官能基藥物對CYP3A4酵素活性的抑制現象-
------------------------------------------------------ 23
三、抗黴菌唑(azole)官能基藥物對CYP3A4 mRNA及蛋白質表現量
的影響------------------------------------------------ 24
四、抗黴菌唑(azole)官能基藥物破壞PXR和SRC-1間交互作用---
------------------------------------------------------ 24
伍、結果歸納與探討------------------------------------ 26
參考文獻---------------------------------------------- 30
附表-------------------------------------------------- 37
附圖-------------------------------------------------- 42
自述-------------------------------------------------- 59
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