臺灣博碩士論文加值系統

苯二氮平(benzodiazepine, BDZ)類藥物濫用在醫療院所以及台灣青少年團體是頗為嚴重的問題，藥物主要結構為5-aryl-1,4-benzodiazepine，屬於硝基多環芳香烴化合物，而氟硝西泮(flunitrazepam, FNZ)為屬於BDZ類藥物的一種，具有安眠、鎮靜、抗焦慮、肌肉鬆弛及抗痙攣等作用，主要用來治療短期失眠症狀或作為催眠藥和麻醉前用藥。此外由於FNZ具有陶醉感作用(euphoric effects)以及短暫失憶的效果，因而常為青少年濫用以及被有心人士加入飲料中迷昏特定人物以達到犯罪的目的。
過去報導某些微生物可以藉由氧化或是還原途徑來降解或是轉換硝基芳香烴化合物來減低毒性，而許多偶氮化合物(azo compound)以及帶硝基(nitro group)的外來物質於生物體內的還原代謝反應主要位於腸道中的腸內菌進行。除此之外FNZ於人體內可以經由氧化代謝作用生成N-desmethylflunitrazepam (N-DF)以及3-hydroxyflunitrazepam (3-HF)，經還原代謝作用則生成7-aminoflunitrazepam (7-AF)。目前研究得知人體細胞色素P450 CYP 2C19及CYP 3A4為主要參與FNZ氧化代謝反應產生N-DF及3-HF，而本實驗室過去研究發現除了肝臟NADPH-cytochrome P450 reductase可以參與FNZ的還原反應，也發現腸、及腸道中的腸內菌可以參與FNZ的還原代謝反應，而腸道菌株經有氧培養下得到三種腸內菌株Escherichia coli、salmonella typhi以及enterobacter cloacae，其中並證明腸內菌Escherichia coli酵素硝基還原酶nfsB對FNZ的還原作用的參與。因而引發本篇實驗研究動機探討腸道內菌株Escherichia coli硝基還原酶nfsA、nfsB以及ydjA對於FNZ還原反應酵素動力學相關的探討以及硝基還原酶胺基酸序列點突變對於硝基還原酶還原代謝FNZ能力之影響。
實驗設計首先驗證腸道內菌株Escherichia coli、Salmonella typhi以及Enterobacter cloacae細胞溶質蛋白質對於FNZ還原代謝之活性並確認硝基還原酶蛋白質以及硝基還原酶nfsA、nfsB以及ydjA同源基因的表現，並以基因轉殖技術得到純化後的硝基還原酶nfsA、nfsB以及ydjA來探討其對FNZ之酵素還原活性，此外也對硝基還原酶nfsB中酵素受質-活性結合位置作胺基酸序列點突變取代試驗，並以酵素蛋白質立體結構的角度來探討胺基酸序列點突變對於硝基還原酶酵素受質-活性結合位置與FNZ還原代謝能力的影響。
結果發現三種腸道內菌株細胞溶質皆可以對FNZ進行還原代謝反應，並有硝基還原酶蛋白質以及同源基因nfsA、nfsB以及ydjA之messenger RNA表現；此外Escherichia coli硝基還原酶nfsA以及NfsB皆可以對FNZ進行還原代謝反應生成代謝物7-aminoflunitrazepam，而硝基還原酶ydjA完全無法對FNZ進行還原反應；在酵素動力學探討硝基還原酶nfsB胺基酸序列點突變對酵素活性之影響，發現點突變Asn-71-Ser與Phe-124-Trp相較於野生型其酵素催化常數(Kcat/Km)分別提升約2以及5倍左右。综合以上結果，認為腸道內菌株硝基還原酶可以參與FNZ之還原代謝作用並且對受質可能具有選擇的特異性，此外硝基還原酶酵素受質-活性結合位置內胺基酸序列點突變Asn-71-Ser與Phe-124-Trp可以幫助提升酵素的代謝速率，並期待其有助於幫助未來應用於人體flunitrazepam藥物濫用之解毒劑。

Benzodiazepine (BDZ) abuse in hospitals and by young people is a growing problem and carries serious risks to health and society. BDZ are a class of psychoactive drugs with varying hypnotic, sedative, anxiolytic, anticonvulsant, muscle relaxant and amnesic properties, which are mediated by slowing down the central nervous system. Flunitrazepam (FNZ) is a short-intermediate acting BDZ derivative, prescribed for the treatment of insomnia, marketed by Roche most commonly under the trade name Rohypnol, has a high affinity for the BDZ receptor within the gama-aminobutyric acid (GABA) complex. In humans, FNZ is oxidized to the major metabolites N-demethylflunitrazepam (NDF) and 3-hydroxyflunitrazepam (3HF) and reduced to 7-aminoflunitrazepam (7AF). Human CYP2C19 and CYP3A4 are the principal P-450 cytochromes involved in NDF and 3HF formation. In our laboratory previous researches, we know not only the liver NADPH-cytochrome P450 reductase, but also intestinal micorflora are involved in 7AF formation. Furthermore, we obtained the three microflora form intestine: Escherichia coli, Salmonella typhi, and Enterobacter cloacae under aerobic culture condition, and confirmed that Escherichia coli nitroreductase nfsB involved in FNZ reduction for 7AF formation. In order to figure out intestinal micorflora nitroreductase in FNZ reductive metabolism, it therefore seemed worth to investigate the role of E. coli nitroreductases in the reduction of FNZ.
In this study, first we confirmed the activities of FNZ reductive reaction by cytosol proteins of intestinal microflora Escherichia coli, Salmonella typhi, and Enterobacter cloacae, and then made sure the nitroreductase activities in cytosol proteins and messenger RNA express levels in homologous genes nfsA, nfsB, and ydjA. Secondary, used the purified E. coli nitroreductases nfsA, nfsB, and ydjA for FNZ reductive analysis. Finally, we did the amino acid sequence point mutations in nitroreductase nfsB for kinetic studies, and combined the protein structures to investigate the effects of point mutation for enzyme activity.
The following results were obtained: (I) the activities of 7-AF formation, protein nfsB, and mRNA of homologous genes nfsA, nfsB, ydjA could be observed in all the cytosol proteins of three intestinal microflora; (II) the E. coli nitroreductases nfsA and nfsB both could metabolite FNZ for 7-AF formation, but not to ydjA; (III) point mutations of Asn-71-Ser, and Phe-124-Trp could enhance the enzyme catalytic constant (Kcat/Km).
Therefore, these results suggested that the nitroreductases of intestinal microflora were involved in FNZ reduction, and supposed that they may have selective specificity for substrates. In addition, the kinetic data of point mutations showed the point mutants Asn-71-Ser, and Phe-124-Trp could enhance the enzyme activity for 7AF formation. In future, we expect to find the high efficiency nitroreductase would be applied to antidote for flunitrazepam abuse.

第一章 緒言 1
1.1 苯二氮平類藥物 1
1.2 氟硝西泮 2
1.3 硝基芳香烴化合物的還原代謝反應 4
1.4 硝基還原酶 5
1.5 腸內菌對外來物質代謝的角色 7
1.6 酵素受質-活性結合位置的研究 9
1.7 研究動機與目的 11
第二章 實驗材料與方法 13
2.1 實驗藥品 13
2.2 實驗器材 14
2.3 實驗菌株 15
2.4 實驗方法 16
第三章 實驗結果 25
3.1 腸內菌HPLC分析flunitrazepam及其代謝物7-aminoflunitrazepam 25
3.2 Flunitrazepam及其代謝物7-aminoflunitrazepam之HPLC標準曲線及線性回歸公式 …………………………………………………………………………………………………………………………..25
3.3 HPLC分析方法精密度(precision)與準確度(accuracy)之分析 26
3.4 腸道內菌株Escherichia coli、Salmonella typhi以及Enterobacter cloacae細胞溶質對flunitrazepam之代謝活性 26
3.5 以西方點墨法分析腸道菌株Escherichia coli、Salmonella typhi以及Enterobacter cloacae細胞溶質中硝基還原酶nfsB的表現 27
3.6 以逆轉錄酶鏈鎖反應分析腸道菌株Escherichia coli、Salmonella typhi以及Enterobacter cloacae硝基還原酶nfsA、nfsB以及ydjA的基因表現層級 27
3.7 探討Escherichia coli硝基還原酶nfsA、nfsB以及 ydjA對flunitrazepam還原反應酵素之活性 28
3.8 探討硝基還原酶nfsB胺基酸序列點突變對於flunitrazepam還原反應酵素活性之影響 …………………………………………………………………………………………………………………………..28
3.9 探討時間效應對硝基還原酶nfsA、nfsB以及nfsB點突變型於flunitrazepam還原反應酵素活性之影響 29
3.10 硝基還原酶nfsA、nfsB以及nfsB點突變型對於flunitrazepam還原反應酵素酵素動力學之探討 30
第四章 討論 31
4.1 HPLC分析flunitrazepam以及其還原代謝產物7-aminoflunitrazepam模式的建立與探討………………………………………………………………………………………………………………………..31
4.2.1 腸道內菌株Escherichia coli、Salmonella typhi以及Enterobacter cloacae硝基還原酶對於flunitrazepam還原代謝反應的探討 32
4.2.2 腸道內菌株Escherichia coli硝基還原酶nfsA、nfsB以及ydjA對於flunitrazepam還原代謝酵素活性的探討 34
4.3 腸道內菌株Escherichia coli硝基還原酶nfsB胺基酸序列點突變對酵素受質-活性結合位置影響以及酵素動力學的探討 36
4.4 總結與未來展望 39
參考文獻 41
圖表集 47
Appendix A. supplementary data 59

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