大腸桿菌中氧氣不敏感性之硝基還原酶NfsB對Flunitrazepam的解毒作用之新的展望

Abstract

苯二氮平 (benzodiazepines, BDZ) 是一類臨床常用的中樞神經系統抑制藥物，而氟硝西泮 (flunitrazepam, FZ) 為BDZ類藥物中的一種，商品名是Rohypnol (瑞士羅氏藥廠產)，FZ化學名稱為5-(2-fluorophenyl)-1,3,dihydro-1-methyl-7-nitro-2H-1,4- benzodiazepin-2-one，在化學結構中因為具有硝基及氟原子使其具有安眠、鎮靜、抗焦慮和肌肉鬆弛等作用，在分類上FZ屬長效型BDZ，在台灣FZ必須經由醫師開立處方才可取得。但由於服用後具有陶醉感作用(euphoric effects)，使其成為年輕人使用的濫用藥物之一，並被廣泛使用為犯罪工具，因此又被稱為”約會強暴藥” (date-rape drug) ，在台灣一般人稱為FM2。FZ於人體內可經由氧化作用生成N-desmethylflunitrazepam (N-DFZ) 以及3-hydroxyflunitrazepam (3-HFZ) ，或經還原作用生成7-amino-flunitrazepam (7-AFZ)。 本實驗室先前研究發現肝臟NADPH-cytochrome P450 reductase可參與FZ的還原反應，以及發現腸內菌中硝基還原酶也可在腸道中將FZ進行還原作用。文獻報導FZ及N-DFZ具安眠作用，FZ及其代謝物90%經由尿液排出，10%經由糞便排出。過去臨床上曾使用Flumazenil (1,4-Midazobenzodiazepine)，亦屬BDZ類物質，結構上同樣具有1,4-diazepine，可透過與FZ的競爭性阻斷作用來達到解毒目的，但因為Flumazenil藥效短，在急診使用Flumazenil並不能減少這些病人的罹病率及死亡率，且對於使用Flumazenil後清醒之病人需小心觀察至少十二小時，以免因效果減退，病人再度昏迷或進一步發生呼吸抑制等危險現象，所以若用於維持性治療時劑量往往需隨時依病患之反應作調整，不甚方便實際，甚至危險。因而激發本研究動機，即如何加速7AFZ產生及排除率的增加，降低FZ及N-DFZ在體內的累積量。實驗設計以in vitro及in vivo方式進行。首先以硝基還原酶NfsB野生型分別在有氧及厭氧環境中進行FZ還原代謝反應。接著再以基因轉殖技術，將E.coli中硝基還原酶NfsB其酵素受質-活性結合位置在胺基酸序列Tyr-68-Gly、Phe-70-His、Asn-71-Ser及Phe-124-Trp等位置進行點突變，反應結果發現單點突變型還原代謝作用皆比野生型有顯著上增加。再以最佳代謝結果的突變位置Asn-71-Ser及Phe-124-Trp進行雙突變後做酵素動力學之探討，結果顯示雙點突變型更能有效提升還原代謝作用。此外，酵素與基質間親和力以野生型為最佳，若以一定量的酵素所能催化的最高反應速率則以雙點突變型最高。最後透過動物模式了解硝基還原酶NfsB及經點突變之硝基還原酶對小鼠處理FZ的毒性之探討，利用組織病理切片觀察腦部、心臟、肺臟、脾臟、肝臟及腎臟，亦透過血清及尿液臨床生化值測定，評估認為硝基還原酶對小鼠生物體並無顯著傷害。利用ELISA方式檢測硝基還原酶NfsB及經點突變之硝基還原酶在小鼠之酵素動力學，結果發現NfsB野生型在小鼠體內半衰期約為6.85小時且單點突變型與雙點突變型半衰期分別更長達至14.14與28.91小時。同時測量硝基還原酶NfsB及經點突變之硝基還原酶對小鼠處理FZ後，在6小時的尿液及12小時的血清中，所測得之FZ消耗量皆以雙點突變型最多。最後記錄硝基還原酶NfsB及經點突變之硝基還原酶對小鼠處理FZ睡眠時間改變之觀察，結果顯示，單點突變型可明顯縮短睡眠時間，而雙點突變型更可縮短將近一半睡眠時間，綜合實驗各項結果，提供未來硝基還原酶NfsB實際應用在人體中當作對FZ的解毒劑新的展望。

Benzodiazepines (BDZ) are clinical drugs that slow certain types of nerve signals throughout the central nervous system. Flunitrazepam (FZ), Rohypnol®, 5-(2-fluorophenyl)-1,3-dihydro-1- methyl-7-nitro-2H-1,4-benzodiazepin-2-one, which has a nitro group and a fluorine atom in the molecule, is a benzodiazepine derivative and pharmacologically a full agonist whose hypnotic effect predominates over the sedative, anxiolytic, muscle relaxing and anticonvulsant effects, characteristics of benzodiazepines. FZ, a long-acting benzodiazepine, is a prescription drug in Taiwan. Unfortunenately, for its euphoric effect, FZ is a popular drug abused among young adults, and often used by rapists, therefore FZ has been nicknamed as “date-rape drug”. In Taiwan, it is also called FM2. In human, FZ is oxidized to the major metabolites N-demethylflunitrazepam (N-DFZ), 3-hydroxyflunitrazepam (3-HFZ) and reduced to 7-aminoflunitrazepam (7-AFZ). In previous study from our lab, we demonstrated not only NADPH- cytochrome P450 reductase in liver but also nitroreductase of the intestinal microflora were involved in FZ reduction. According to other studies, FZ and N-DFZ have sedative effect. About 90 % of a single dose is eliminated by the kidney after biotransformation, while about 10 % is eliminated in the feces. In the past, Flumazenil, which also has 1,4-diazepine, was a specific benzodiazepine antagonist which can prevent or abolish selectively at the receptor level all centrally mediated effects of benzodiazepines, but the duration of benzodiazepine-antagonistic action of flumazenil is limited. In emergency case, Flumazenil can not reduce the mortality of the patients, furthermore, the flumazenil infusion was necessary to keep the patient fully awake and able to cough. Therefore, unless additional information becomes available supporting flumazenil's ability to reverse benzodiazepine-induced respiratory depression, it should not be used for this indication. However, the aim of the present experiment is how to increase the formation of 7-AFZ and the velocity of elimination, and to decrease the accumulation of FZ and N-DFZ. It can be separated in vitro and in vivo parts. First of all, the experiment confirmed the enzyme activity of FZ reduction by nitroreductase NfsB under anaerobic and aerobic condition. Secondary, we did the amino acid sequence point mutants including Tyr-68-Gly, Phe-70-His, Asn-71-Ser and Phe-124-Trp, the enzyme-substrate active site in nitroreductase NfsB and to proceeded reductive reaction. We found that the point mutants Asn-71-Ser and Phe-124-Trp have the two of the best enzyme activity for 7-AFZ formation, thus to be the double mutant then compared with wild type and single mutant type. From its kinetic study, we found that double mutant enhance more reductive reaction. About the affinity between enzyme and substrate, wild type is the best. However, the double mutant has the highest Vmax value. Finally, we found out the toxicity of FZ after injection of nitroreductase NfsB and its mutants by tissue pathology including brain, heart, lung, spleen, liver and kidney, and by biochemical data of serum and urine to assess nitroreductase in mice is safety without significant injury. We found that the kinetic were performed with nitroreductase NfsB and its mutants in mice by ELISA assay. In mice, the half life of nitroreductase NfsB wild type was 6.85 hours and its single and double mutant was even more to 14.14 and 28.91 hours, respectively. In addition, the consumption of FZ in urine at 6 hours, and in serum at 12 hours, double mutant had the greatest efficiency. Finally, we recorded the sleeping time recovery experiments. The sleeping time was also significant reduce by single point mutant, moreover, the double mutant could reduce half sleeping time. Therefore, these results suggested that nitroreductase NfsB could be the new perspective on the detoxification of FZ in human in the future.