In Vitro Metabolic Study of Temsirolimus: Preparation, Isolation, and Identification of the Metabolites
In Vitro Metabolic Study of Temsirolimus: Preparation, Isolation, and Identification of the Metabolites
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DOI：
10.1124/dmd.107.014746
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发表时间：
2007-09
影响因子：
3.9
通讯作者：
P. Cai;R. Tsao;M. Ruppen
中科院分区：
文献类型：
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作者：
P. Cai;R. Tsao;M. Ruppen
使用人肝微粒体以及重组人细胞色素P450（即CYP 3A 4、1A 2、2A 6、2C 8、2C 9、2C 19和2 E1）研究了替西罗莫司（雷帕霉素-42-[2，2-双-（羟甲基）]-丙酸酯）的体外代谢。通过液相色谱（LC）-串联质谱（MS/MS或MS/MS/MS）检测到15种代谢物。CYP 3A 4被确定为负责该化合物代谢的主要酶。替西罗莫司与重组CYP 3A 4孵育产生了与人肝微粒体孵育检测到的大部分代谢产物，用于大规模制备代谢产物。通过硅胶色谱法，然后半制备反相高效液相色谱法，单独的代谢产物进行分离和纯化的结构解析和生物活性研究。通过正负极质谱（MS）和MS/MS光谱方法，将次要代谢产物（峰1-7）鉴别为羟基化或去甲基化大环内酯开环替西罗莫司衍生物。由于这些化合物不稳定且仅以痕量存在，因此未进行进一步研究。采用LC-MS、MS/MS、MS/MS/MS和NMR技术，鉴别了6种主要代谢产物，分别为36-羟基坦西罗莫司（M8）、35-羟基坦西罗莫司（M9）、具有开放半缩酮环的11-羟基坦西罗莫司（M10和M11）、N-氧化坦西罗莫司（M12）和32-O-去甲基坦西罗莫司（M13）。与母体化合物相比，这些代谢产物对LNCaP细胞增殖的活性显著降低。
The in vitro metabolism of temsirolimus, (rapamycin-42-[2,2-bis-(hydroxymethyl)]-propionate), an antineoplastic agent, was studied using human liver microsomes as well as recombinant human cytochrome P450s, namely CYP3A4, 1A2, 2A6, 2C8, 2C9, 2C19, and 2E1. Fifteen metabolites were detected by liquid chromatography (LC)-tandem mass spectrometry (MS/MS or MS/MS/MS). CYP3A4 was identified as the main enzyme responsible for the metabolism of the compound. Incubation of temsirolimus with recombinant CYP3A4 produced most of the metabolites detected from incubation with human liver microsomes, which was used for large-scale preparation of the metabolites. By silica gel chromatography followed by semipreparative reverse-phase high-performance liquid chromatography, individual metabolites were separated and purified for structural elucidation and bioactivity studies. The minor metabolites (peaks 1-7) were identified as hydroxylated or desmethylated macrolide ring-opened temsirolimus derivatives by both positive and negative mass spectrometry (MS) and MS/MS spectroscopic methods. Because these compounds were unstable and only present in trace amounts, no further investigations were conducted. Six major metabolites were identified as 36-hydroxyl temsirolimus (M8), 35-hydroxyl temsirolimus (M9), 11-hydroxyl temsirolimus with an opened hemiketal ring (M10 and M11), N- oxide temsirolimus (M12), and 32-O-desmethyl temsirolimus (M13) using combined LC-MS, MS/MS, MS/MS/MS, and NMR techniques. Compared with the parent compound, these metabolites showed dramatically decreased activity against LNCaP cellular proliferation.