Identification of enzymes responsible for nitrazepam metabolism and toxicity in human
Identification of enzymes responsible for nitrazepam metabolism and toxicity in human
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负责人体硝西泮代谢和毒性的酶的鉴定
DOI：
10.1016/j.bcp.2017.06.114
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发表时间：
2017
影响因子：
5.8
通讯作者：
Nakajima Miki
中科院分区：
文献类型：
--
作者：
Konishi Keigo;Fukami Tatsuki;Gotoh Saki;Nakajima Miki
硝西泮（NZP）是一种催眠药，很少引起人类肝损伤和啮齿动物致畸性。在人体中，NZP主要通过还原代谢为7-氨基硝西泮（ANZP），随后通过乙酰化代谢为7-乙酰氨基硝西泮（AANZP）。在啮齿动物中，ANZP可以通过水解从AANZP中再生，但尚不清楚这种反应是否发生在人类中。在啮齿类动物中，AANZP可能与致畸性相关，而在人类中，已知药物诱导的肝损伤可能由NZP反应性代谢物引起。在这项研究中，我们试图确定负责NZP代谢的酶，以获得对该过程和相关代谢物毒性的基本了解。我们发现人肝细胞质（HLC）中的NZP还原酶活性高于人肝微粒体（HLM）。我们从HLC中纯化了负责酶，发现NZP还原酶是醛氧化酶1（AOX 1）。AOX 1的作用通过观察到添加AOX 1的电子供体N1-甲基烟酰胺后NZP还原酶活性的增加以及AOX 1抑制剂存在下HLC中该活性的抑制来证实。ANZP经N-乙酰基转移酶（NAT）2乙酰化形成AANZP。利用重组酯酶进行抑制实验，发现AANZP在人肝中被芳基乙酰胺脱乙酰酶（AADAC）水解，通过NZP还原和ANZP羟基化反应，检测到N-羟基氨基NZP与N-乙酰-L-半胱氨酸结合，推测其为活性代谢产物。在后一反应中，在检测的各种P450亚型中，重组CYP 3A 4很容易形成结合物。总之，我们发现AOX 1、NAT 2、AADAC和CYP 3A 4是NZP药代动力学的决定因素，它们赋予NZP副作用敏感性的个体间差异。
Nitrazepam (NZP) is a hypnotic agent that rarely causes liver injuries in humans and teratogenicity in rodents. In humans, NZP is primarily metabolized to 7-aminonitrazepam (ANZP) by reduction and subsequently to 7-acetylamino nitrazepam (AANZP) by acetylation. ANZP can be regenerated from AANZP by hydrolysis in rodents, but it is still unclear whether this reaction occurs in humans. In rodents, AANZP may be associated with teratogenicity, while in humans, it is known that drug-induced liver injuries may be caused by NZP reactive metabolite(s). In this study, we attempted to identify the enzymes responsible for NZP metabolism to obtain a basic understanding of this process and the associated metabolite toxicities. We found that the NZP reductase activity in human liver cytosol (HLC) was higher than that in human liver microsomes (HLM). We purified the responsible enzyme(s) from HLC and found that the NZP reductase was aldehyde oxidase 1 (AOX1). The role of AOX1 was confirmed by an observed increase in the NZP reductase activity upon addition ofN1-methylnicotinamide, an electron donor of AOX1, as well as inhibition of this activity in HLC in the presence of AOX1 inhibitors. ANZP was acetylated to form AANZP byN-acetyltransferase (NAT) 2. An experiment using recombinant esterases in an inhibition study using HLM revealed that AANZP is hydrolyzed by arylacetamide deacetylase (AADAC) in the human liver.N-Hydroxylamino NZP, which is suspected to be a reactive metabolite, was detected as a conjugate withN-acetyl-l-cysteine through NZP reduction and ANZP hydroxylation reactions. In the latter reaction, the conjugate was readily formed by recombinant CYP3A4 among the various P450 isoforms tested. In sum, we found that AOX1, NAT2, AADAC, and CYP3A4 are the determinants for the pharmacokinetics of NZP and that they confer interindividual variability in sensitivity to NZP side effects.