臺灣博碩士論文加值系統

本研究首先依據本實驗室開發的製程模式，利用液相層析技術 (HPLC)及電噴灑質譜儀 (ESI-MS)，製備1,4-dihydropyridine (DHP) 類降血壓藥物Nifedipine (NF) 及Nisoldipine (NS) 與維生素C在光照過程中產生的新穎化合物6-Hydroxy-2,4-dimethyl-5-oxo-5,6-dihydrobenzo[c][2,7]naphthyridinen-1-carboxylic acid methyl ester (cpd.4)及6-Hydroxy-2,4-dimethyl-5-oxo-5,6-dihydrobenzo[c][2,7]naphthyridinen-1-carboxylic acid isobutyl ester (cpd.8)。
第一部分使用XTT assay 測定cpd.4及cpd.8對大鼠平滑肌A7r5 cell的細胞毒性，實驗結果得知cpd.4及cpd.8對A7r5 cell的毒性是相當低的，因此我們認為NF及NS與維生素C在光照環境下反應所生成的cpd.4及cpd.8對動物細胞沒有毒性為害；實驗第二部分探討cpd.4及cpd.8在A7r5 cell中阻斷鈣離子通道能力的改變，分別使用流式細胞儀、雷射共軛焦掃描顯微鏡及Calcium Colorimetric Assay Kit三種方法來相互佐證，由實驗結果得知cpd.4及cpd.8在阻斷鈣離子進入細胞的能力上皆具有劑量依賴關係，當所使用的濃度越高，阻斷鈣離子的能力越強，然而其降低細胞內鈣離子濃度的幅度大小都相對的比NF及NS低，。
根據以上的實驗結果，假使高血壓患者未將DHP類光敏感型藥物NF及NS妥善保存，將其暴露於光照下，而誤食藥物受光刺激的光解產物後與人體血清及肝組織中的維生素C反應，產生的新化合物雖然對動物細胞不會有毒性的危害，但鈣離子阻斷的能力則會明顯地降低，也就無法在相同劑量上達到有效降血壓作用。

關鍵字：鈣離子阻斷劑、Nifedipine、Nisoldipine、Fluo-3 AM

This study was based on the preparation of new interactive compounds between photosensitive 1,4-dihydropyridine drugs (i.e. nifedipine and nisoldipine) and vitamin C. These compounds are purified and identified as 6-Hydroxy-2,4-dimethyl-5-oxo-5,6-dihydrobenzo[c][2,7]naphthyridinen-1-carboxylic acid methyl ester (named as cpd.4 in this thesis) and 6-Hydroxy-2,4-dimethyl-5-oxo-5,6-dihydrobenzo[c][2,7] naphthyridinen-1-carboxylic acid isobutyl ester (named as cpd.8) using high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS).
In the first part of the thesis, we determine cytotoxicity of cpd.4 and cpd.8 on the A7r5 cells by XTT assay. The test results have confirmed that the cytotoxicity of cpd.4 and cpd.8 on A7r5 cells is quite low. In the second part, we investigated the effect of cpd.4 and cpd.8 on the blocking of calcium ion channels on A7r5 cells, using flow cytometry, confocal laser scanning microscope, and Calcium Colorimetric Assay Kit. The results showed that cpd.4 and cpd.8 are able to decrease intracellular calcium ion levels due to blocking calcium ion channels in a manner of dose dependence. However, the magnitude of cpd. 4 and cpd.8 on blocking the channels is relatively lower in comparison with NF and NS, respectively.
Another message calls, based on the found evidence, hypertensive patients would not have proper medication if NF or NS were not preserved well. These photosensitive drugs will undergo photolysis and interact with vitamin C in human serum and liver tissue to produce the new compounds which could show different pharmacodynamics and pharmacokinetics as expected. To the least extent, the new compounds would cause a decreased ability of blocking calcium channels and less achieve the antihypertensive effect although aren’t harmful to the human body (XTT assay).

目錄........................................... i
圖目錄......................................... iv
表目錄......................................... vi
中文摘要....................................... vii
英文摘要....................................... viii
第一章 緒論................................... 1
1-1藥物光過敏反應及機制.......................... 1
1-1-1藥物光過敏反應............................. 1
1-1-2藥物光過敏機制............................. 1
1-2鈣離子...................................... 3
1-2-1鈣離子濃度對高血壓之影響.................... 4
1-2-2細胞內鈣離子濃度的測量...................... 5
1-3鈣離子阻斷劑................................. 6
1-3-1 Dihydropyridine (DHP).................... 7
1-3-2 Nifedipine (NF).......................... 8
1-3-3 Nisoldipine (NS)......................... 10
1-3抗氧化劑代謝物-維生素 C....................... 11
1-5液相層析技術與質譜分析方法原理................. 13
1-3-1液相層析技術原理........................... 13
1-3-2質譜分析方法原理........................... 14
第二章 研究動機與目的.......................... 15
第三章 實驗材料與方法.......................... 18
3-1實驗材料..................................... 18
3-1-1藥品...................................... 18
3-1-2緩衝液與配方............................... 19
表3-1 Hank’s buffer saline配方.................. 20
3-1-3細胞株..................................... 20
3-1-4細胞培養基................................. 20
3-1-4-1 DMEM................................... 20
3-1-4-2 A7r5 cells growth medium............... 21
3-2實驗儀器與裝置............................... 21
3-3實驗方法..................................... 23
3-3-1 cpd.4及cpd.8的製備........................ 23
3-3-2樣品stock溶液.............................. 24
3-3-3細胞培養................................... 25
3-3-3-1活化冷凍細胞............................. 25
3-3-3-2繼代培養................................. 25
3-3-3-3冷凍細胞................................. 25
3-3-3-4細胞計數................................. 26
3-3-4細胞毒性試驗 (XTT assay)................... 26
3-3-5細胞內鈣離子濃度............................27
3-3-5-1雷射共軛焦掃描顯微鏡...................... 28
3-3-5-2流式細胞儀之應用......................... 29
3-3-5-3 Calcium Colorimetric Assay Kit......... 30
第四章 結果與討論.............................. 32
4-1 cpd.4及cpd.8的鑑定.......................... 32
4-2 cpd.4及cpd.8的細胞毒性試驗.................. 36
4-3細胞內鈣離子濃度............................. 37
4-3-1雷射共軛焦掃描顯微鏡........................ 37
4-3-2流式細胞儀之應用........................... 42
4-3-2-1 NF藥物與cpd.4鈣離子阻斷能力比較.......... 42
4-3-2-2 NS藥物與cpd.8鈣離子阻斷能力比較.......... 47
4-3-2-3 量化螢光強度............................ 51
4-3-3 Calcium Colorimetric Assay Kit........... 53
第五章 結論..................................... 55
第六章 參考文獻................................. 57

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