硝基還原酶參與Flunitrazepam還原代謝作用之探討

The Role of Nitroreductase in the Reductive Metabolism of Flunitrazepam

陳玉蓮(Yu-Lian Chen)

指導教授 ： 彭福佐

國立臺灣大學/醫學院/毒理學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.00838

摘要

Flunitrazeapm (FNZ) 與一般 bezodiazepine 鎮靜劑一樣，都具有解除焦慮、鎮靜、肌肉鬆弛、安眠、抗痙攣等療效。由於FNZ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 。除此之外，FNZ化學結構中具氟原子及硝基，其安眠作用強且藥效持久，因而常被一些有心人士加以利用，加入飲料中迷昏特定人物以達到犯罪目的，所以也有「強暴藥丸」之惡名。
FNZ於人體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)。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。至於那一酵素參與FNZ還原代謝反應目前尚未明瞭。
已有文獻報導許多偶氮化合物 (azo compound) 或含硝基 (nitro group) 之外來物質，其還原代謝反應主要於腸道中之腸內菌進行，而腸內菌能進行外來物質之還原代謝反應，主要是因為腸內菌含有高活性之硝基還原酶 (nitroreductase, NTR) ，因此，本研究的主要目的即在探討腸內菌NTR參與FNZ之還原代謝反應所扮演的角色。
外來物質的代謝與性別及年齡有差異性，甚至於已有報導NTR的表現與性別及年齡有關聯性。因此，本研究也於不同年齡及性別之大鼠中探討FNZ在肝臟、小腸組織以及腸內菌的還原代謝反應。
本研究分別從不同年齡雄性以及雌性大鼠之肝臟、小腸及腸內菌中探討NTR活性、NTR蛋白表現，另外，以化學抑制以及抗體抑制方式以及使用表現NTR之人類卵巢癌細胞株SKOV3NR與純化之大腸桿菌NTR酵素來進一步探討NTR在FNZ還原代謝過程當中所扮演的角色。
實驗結果顯示：(1) FNZ還原成7-AF的主要位置是在腸內菌；雌性大鼠對FNZ之還原代謝能力較雄性大鼠來的高，且在厭氧狀態下7-AF的產生較有氧狀態高；(2) FNZ於8週大雄性大鼠腸內菌之還原代謝反應有劑量及時間效應關係；(3) FNZ在腸內菌以及肝臟的還原代謝作用隨著年齡的增加而增加，小腸組織則隨著年齡增加而減少；(4) 以NTR之化學抑制劑4-NBA以及2-IBA探討其對FNZ還原代謝之抑制作用，結果顯示2-IBA較4-NBA更能有效抑制7-AF的產生；(5) 專一性之NTR抗體也可以有效的抑制7-AF的產生；(6) 利用西方點墨法可以偵測出大鼠肝、小腸以及腸內菌中NTR的表現； (7) 利用西方點墨法可以偵測出SKOV3NR細胞株中NTR的表現； (8) FNZ在大量表現NTR之人類卵巢癌細胞株 (SKOV3NR cell line) 中的還原代謝能力也隨著時間而增加，至48小時達到極限；而以 E. coli.純化之NTR酵素進行代謝反應，也得知隨著濃度的增加，其7-AF的產量也隨之增加，且於厭氧下7-AF的產量較於有氧下高。
综合以上結果，推測腸內菌NTR是參與FNZ還原代謝反應的重要酵素。

關鍵字

還原代謝 ； 氟硝西泮 ； 腸內菌 ； 硝基還原酶

並列摘要

The benzodiazepine flunitrazepam is extensively prescribed to patients with insomnia in many countries, but has also become popular among drug abusers. In Taiwan, flunitrazepam (FNZ) has been prescribed by doctors for controlling hyponic, sedative, anxiolytic, muscle-relaxing and anticonvulsant effects of patients. FNZ has a nitro group and a fluorine atom in the molecule, both of them increase the hypnotic effect of benzodiazepine.
In human, FNZ is oxidized to the major metabolites N-demethylflunitrazepam (NDF), 3-hydroxyflunitrazepam (3HF) and reduced to 7-aminoflunitrazepam (7-AF). It has been identified CYP2C19 and CYP3A4 as the principal cytochrome P450s involved in the metabolism of FNZ to its major metabolites N-DF and 3-HF. However, it is not clear which enzyme is responsible for the reduction of FNZ to 7-AF.
The reduction of certain xenobiotics is also mediated by mixed and pure cultures of intestinal microflora. It therefore seemed worth investigating the role of intestinal microflora in the reduction of FNZ. The overarching goal of this study was to delineate the reductive metabolism of FNZ by intestine microflora.
In addition, it has been reported that reduction of the nitro moiety to the corresponding amine is catalyzed by nitroreductase. Therefore, it led us to propose that nitroreductase is involved in the reductive metabolism of FNZ.
Sex and age differences in the metabolism of xenobiotics are well known. It has been shown that nitroreductse expression was related to age and sex difference. Therefore, in this study, we also studied gender- and age-related changes in FNZ reductive metabolism in Wistar rats.
In order to elucidate the role of nitroreductase in FNZ reductive metabolism, the experiments were performed with SKOV3NR cell line derived from human ovarian carcinoma cell line, in which E. coli. nitroreductase was expressed. Furthermore, the chemical inhibitor 4-nitrobenzoic acid (4-NBA) and 2-iodosobenzoic acid (2-IBA), E. coli. nitroreductase antibody and purified E. coli. nitroreductase enzyme were also used to identify the nitroreductase enzyme responsible for 7-AF formation.
The following are the results obtained：(1) The amount of 7-AF formed from FNZ in intestinal microflora was higher than liver and intestine cytosol from 8-week-old Wistar rats. (2) The FNZ reductive metabolism was dose- and time- dependent in intestinal microflora from 8-week-old Wistar rats. (3) The amount of 7-AF formed from FNZ in 65-week-old Wistar rats was higher than in 1-week-old Wistar rats and also higher in female rats than in male rats. (4) 2-iodosobenzoic acid (2-IBA), a inhibitor of nitroreductase, significantly inhibited 7-AF formation. (5) The anti-nitroreductase anti-body also significantly inhibited 7-AF production. (6) Immunoblotting assay showed that nitroreductase protein was detected in liver, intestine and intestinal microflora cytosol of wistar rats. (7) Immunoblotting assay showed that nitroreductase protein was actually detected in SKOV3NR. (8) The FNZ reductive metabolism was dose- and time- dependent in SKOV3NR cell line and purified E. coli. nitroreductase.
Therefore, these results suggested that intestinal nitroreductase plays an important role in the reductive metabolism pathway from FNZ to 7-AF.

並列關鍵字

Flunitrazepam ； Reductive metabolism ； Intestinal microflora ； nitroreductase

參考文獻

Abernethy, D. R., Greenblatt, D. J., and Shader, R. I. (1986) Benzodiazepine hypnotic metabolism: drug interactions and clinical implications. Acta Psychiatr Scand Suppl. 332: 32-38.

Ackerley, D. F., Gonzalez, C. F., Keyhan, M., Blake, R., and Matin, A. (2004) Mechanism of chromate reduction by the Escherichia coli protein, NfsA, and the role of different chromate reductases in minimizing oxidative stress during chromate reduction. Environ Microbiol. 6: 851- 860.

Aguilar, E. G., Koldobsky, C., de, Toranzo, E. G., and Castro, J. A. (1987) Species and sex differences in the liver microsomal nitroreductive biotransformation of nifurtimox and benznidazole. Arch Int Pharmacodyn Ther. 287: 181-187.

Amrein, R. (1978) The pharmacokinetics and metabolism of flunitrazepam. Klin Anasthesiol Intensivther. 17: 8-24.

Angermaier, L., and Simon, H. (1983) On nitroaryl reductase activities in several Clostridia. Hoppe Seylers Z Physiol Chem. 364: 1653–1663.