Men Lei, Zhao Yunli, Lin Hongli, Yang Mingjing, Liu Hui, Tang Xing, Yu Zhiguo
School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, 110016, China; Department of Food Analysis, Dalian Ocean School, 40 Linghe Street, Dalian, 116023, China.
Rapid Commun Mass Spectrom. 2014 Oct 30;28(20):2162-70. doi: 10.1002/rcm.7003.
TM-2 (13-(N-Boc-3-i-butylisoserinoyl-4,10-β-diacetoxy-2-α-benzoyloxy-5-β,20-epoxy-1,13-α-dihydroxy-9-oxo-19-norcyclopropa[g]tax-11-ene) is a novel semi-synthetic taxane derivative. Our previous study demonstrated that it is a promising taxane derivative. The in vitro comparative metabolic profile of a drug between animals and humans is a key issue that should be investigated at early stages of drug development to better select drug candidates. In this study, the in vitro metabolic pathways of TM-2 in rat, dog and human liver microsomes were established and compared.
TM-2 was incubated with liver microsomes in the presence of NADPH. Two different types of mass spectrometers - a hybrid linear trap quadrupole orbitrap (LC/LTQ-Orbitrap) mass spectrometer and a triple-quadrupole tandem mass spectrometer (LC/QqQ) were employed to acquire structural information of TM-2 metabolites. Accurate mass measurement using LC/LTQ-Orbitrap was used to determine the accurate mass data and elemental compositions of metabolites thereby confirming the proposed structures of the metabolites. For the chemical inhibition study, selective P450 inhibitors were added to incubations to initially characterize the cytochrome P450 (CYP) enzymes involved in the metabolism of TM-2.
A total of 12 components (M1-M12) were detected and identified as the metabolites of TM-2 in vitro. M1-M5 were formed by hydroxylation of the taxane ring or the lateral chain. Hydroxylated products can be further oxidized to the dihydroxylated metabolites M6-M10. M11 was a trihydroxylated metabolite. M12 was tentatively identified as a carboxylic acid derivative. The metabolism of TM-2 is much the same in all three species with some differences. The chemical inhibition study initially demonstrated that the formation of M2, the major metabolite of TM-2, is mainly mediated by CYP3A4.
Hydroxylation is the major biotransformation of the TM-2 pathway in vitro. CYP3A4 may play a dominant role in the formation of M2 in liver microsomes. The knowledge of the metabolic pathways of TM-2 is important to support further research of TM-2.
TM - 2（13 -（N - 叔丁氧羰基 - 3 - 异丁基异丝氨酰基 - 4,10 - β - 二乙酰氧基 - 2 - α - 苯甲酰氧基 - 5 - β,20 - 环氧 - 1,13 - α - 二羟基 - 9 - 氧代 - 19 - 降环丙[a]紫杉 - 11 - 烯）是一种新型半合成紫杉烷衍生物。我们之前的研究表明它是一种有前景的紫杉烷衍生物。药物在动物和人类之间的体外比较代谢概况是药物研发早期应研究的关键问题，以便更好地筛选候选药物。在本研究中，建立并比较了TM - 2在大鼠、犬和人肝微粒体中的体外代谢途径。
在NADPH存在的情况下，将TM - 2与肝微粒体一起孵育。使用两种不同类型的质谱仪——混合线性阱四极杆轨道阱（LC/LTQ - Orbitrap）质谱仪和三重四极杆串联质谱仪（LC/QqQ）来获取TM - 2代谢物的结构信息。使用LC/LTQ - Orbitrap进行精确质量测量，以确定代谢物的精确质量数据和元素组成，从而确认所提出的代谢物结构。对于化学抑制研究，将选择性P450抑制剂添加到孵育体系中，初步鉴定参与TM - 2代谢的细胞色素P450（CYP）酶。
总共检测并鉴定出12种成分（M1 - M12）为TM - 2的体外代谢物。M1 - M5是由紫杉烷环或侧链羟基化形成的。羟基化产物可进一步氧化为二羟基化代谢物M6 - M10。M11是一种三羟基化代谢物。M12初步鉴定为羧酸衍生物。TM - 2在所有三种物种中的代谢基本相同，但存在一些差异。化学抑制研究初步表明，TM - 2的主要代谢物M2的形成主要由CYP3A4介导。
羟基化是体外TM - 2途径的主要生物转化方式。CYP3A4可能在肝微粒体中M2的形成中起主导作用。TM - 2代谢途径的知识对支持TM - 2的进一步研究很重要。
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