In vitro and in vivo glucuronidation of midazolam in humans
In vitro and in vivo glucuronidation of midazolam in humans
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DOI：
10.1111/j.1365-2125.2009.03386.x
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发表时间：
2009-04-01
影响因子：
3.4
通讯作者：
Jones, Barry
中科院分区：
文献类型：
--
作者：
Hyland, Ruth;Osborne, Toby;Jones, Barry
关于该受试者的已知信息中心点咪达唑仑被广泛代谢，并主要以1 '-羟基咪达唑仑结合物的形式在尿液中消除。中心点咪达唑仑已知在体外进行N-葡萄糖醛酸化（通过UGT1A4.center）。在体外，1'-羟基咪达唑仑被UGT 2B 4和UGT 2B 7 O-葡萄糖醛酸化，并被UGT 1A 4 N-葡萄糖醛酸化。本研究补充内容已在人尿液中定量咪达唑仑的中心点N-葡萄糖醛酸，在www.example.com.dot中首次指出了这种代谢途径的发生vivo.center中对4-羟基咪达唑仑的代谢与1 ′-羟基咪达唑仑的代谢进行了比较vitro.center该研究提供了进一步的体外和体内证据，证明了N-葡萄糖醛酸化在咪达唑仑及其代谢物的代谢中的重要性。咪达唑仑（MDZ）是一种苯二氮卓类药物，在临床和体外研究中用作CYP 3A 4探针。已在体外人肝微粒体（HLM）孵育中鉴别出MDZ的葡糖苷酸代谢产物。本研究的主要目的是了解这一途径的体内相关性。通过微粒体孵育产生了N-葡萄糖醛酸苷的真实标准品，并使用固相萃取分离。用质子核磁共振（NMR）和（1）H-（13）C长程相关实验证实了其结构。在人体尿样中对代谢物进行体内定量。在HLM和重组UGT（rUGT）酶中研究了该途径的酶动力学行为。此外，使用1 '-OH咪达唑仑（1'-OH MDZ）和4-OH-咪达唑仑（4-OH MDZ）进行了初步实验以研究N-葡萄糖醛酸化。核磁共振数据证实咪达唑仑N-葡萄糖醛酸（MDZG）标准品在咪唑环的α-氮上缀合。在体内，尿液中的MDZG占给药剂量的1-2%。体外孵育证实UGT 1A 4为目标酶。该途径表现出非典型动力学，应用底物抑制协同结合模型分别测定HLM和rUGT 1A 4中的K（m）（46 μ M，64 μ M）、V（max）（445 pmol min（-1）mg（-1），427 pmol min（-1）mg（-1））和K（i）（58 μ M，79 μ M）。通过与HLM和rUGT酶的孵育，还推断了1 '-OH MDZ和4-OH MDZ的N-葡萄糖醛酸化。MDZ的直接N-葡萄糖醛酸化发生在体内。使用Simcyp（TM）的药代动力学建模表明，在CYP 3A抑制条件下，UGT 1A 4的作用增加。
WHAT IS ALREADY KNOWN ABOUT THIS SUBJECTcenter dot Midazolam is extensively metabolized and is eliminated in urine predominantly as conjugates of 1'-hydroxymidazolam.center dot Midazolam is known to undergo N-glucuronidation in vitro by UGT1A4.center dot In vitro 1'-hydroxymidazolam is O-glucuronidated by UGT2B4 and UGT2B7, and N-glucuronidated by UGT1A4.WHAT THIS STUDY ADDScenter dot N-glucuronide of midazolam has been quantified in human urine, indicating for the first time that this route of metabolism occurs in vivo.center dot Metabolism of 4-hydroxymidazolam has been compared with that of 1'-hydroxymidazolam in vitro.center dot This study provides further evidence, in vitro and in vivo, of the importance of N-glucuronidation in the metabolism of midazolam and its metabolites.Midazolam (MDZ) is a benzodiazepine used as a CYP3A4 probe in clinical and in vitro studies. A glucuronide metabolite of MDZ has been identified in vitro in human liver microsome (HLM) incubations. The primary aim of this study was to understand the in vivo relevance of this pathway.An authentic standard of N-glucuronide was generated from microsomal incubations and isolated using solid-phase extraction. The structure was confirmed using proton nuclear magnetic resonance (NMR) and (1)H-(13)C long range correlation experiments. The metabolite was quantified in vivo in human urine samples. Enzyme kinetic behaviour of the pathway was investigated in HLM and recombinant UGT (rUGT) enzymes. Additionally, preliminary experiments were performed with 1'-OH midazolam (1'-OH MDZ) and 4-OH-midazolam (4-OH MDZ) to investigate N-glucuronidation.NMR data confirmed conjugation of midazolam N-glucuronide (MDZG) standard to be on the alpha-nitrogen of the imidazole ring. In vivo, MDZG in the urine accounted for 1-2% of the administered dose. In vitro incubations confirmed UGT1A4 as the enzyme of interest. The pathway exhibited atypical kinetics and a substrate inhibitory cooperative binding model was applied to determine K(m) (46 mu M, 64 mu M), V(max) (445 pmol min(-1) mg(-1), 427 pmol min(-1) mg(-1)) and K(i) (58 mu M, 79 mu M) in HLM and rUGT1A4, respectively. From incubations with HLM and rUGT enzymes, N-glucuronidation of 1'-OH MDZ and 4-OH MDZ is also inferred.A more complete picture of MDZ metabolism and the enzymes involved has been elucidated. Direct N-glucuronidation of MDZ occurs in vivo. Pharmacokinetic modelling using Simcyp (TM) illustrates an increased role for UGT1A4 under CYP3A inhibited conditions.