Sun Hua, Ma Zhiguo, Lu Danyi, Wu Baojian
Division of Pharmaceutics, College of Pharmacy, Jinan University, Guangzhou, 510632, China.
J Pharm Sci. 2015 Jul;104(7):2369-77. doi: 10.1002/jps.24464. Epub 2015 May 4.
In this study, we aimed to determine the glucuronidation potential of psoralidin in humans and to perform validation on use of psoralidin-3-O-glucuronidation as a functional marker for UGT1A9. Glucuronidation kinetics was determined using human liver microsomes (HLMs), human intestine microsomes (HIM), and expressed UDP-glucuronosyltransferase (UGT) enzymes. The chemical structures of metabolites were determined by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy analyses. Validation of psoralidin-3-O-glucuronidation as a UGT1A9 marker was performed using combined approaches including reaction phenotyping, chemical inhibition, activity correlation analysis, and determination of relative activity factor (RAF). HLM and UGT1A9 generated two monoglucuronides (9-O-glucuronide and 3-O-glucuronide) from psoralidin, whereas HIM, UGT1A1, UGT1A7, and UGT1A8 generated one only (9-O-glucuronide). Formation of 3-O-glucuronide in HLM was markedly inhibited by the UGT1A9-selective inhibitors magnolol and niflumic acid. Further, psoralidin-3-O-glucuronidation was strongly correlated with propofol-glucuronidation in a group of nine individual HLMs (r = 0.978, p < 0.001). Strong correlation was also observed between psoralidin-3-O-glucuronidation and the UGT1A9 protein levels measured by Western blotting (r = 0.944, p < 0.001). Moreover, UGT1A9 was responsible for 99.6% of psoralidin-3-O-glucuronidation in HLM based on the RAF approach. In conclusion, psoralidin was subjected to efficient glucuronidation, generating one or two monoglucuronides depending on UGT isozymes. Also, psoralidin-3-O-glucuronidation was an excellent in vitro marker for UGT1A9.
在本研究中，我们旨在确定补骨脂素在人体内的葡萄糖醛酸化潜力，并对使用补骨脂素-3-O-葡萄糖醛酸化作为UGT1A9的功能标志物进行验证。使用人肝微粒体（HLM）、人肠微粒体（HIM）和表达的尿苷二磷酸葡萄糖醛酸基转移酶（UGT）酶来确定葡萄糖醛酸化动力学。通过液相色谱-质谱联用和核磁共振光谱分析确定代谢物的化学结构。使用包括反应表型分析、化学抑制、活性相关性分析和相对活性因子（RAF）测定在内的联合方法对补骨脂素-3-O-葡萄糖醛酸化作为UGT1A9标志物进行验证。HLM和UGT A9从补骨脂素生成了两种单葡萄糖醛酸苷（9-O-葡萄糖醛酸苷和3-O-葡萄糖醛酸苷），而HIM、UGT1A1、UGT1A7和UGT1A8仅生成一种（9-O-葡萄糖醛酸苷）。UGT1A9选择性抑制剂厚朴酚和尼氟酸显著抑制了HLM中3-O-葡萄糖醛酸苷的形成。此外，在一组九个个体的HLM中，补骨脂素-3-O-葡萄糖醛酸化与丙泊酚葡萄糖醛酸化密切相关（r = 0.978，p < 0.001）。通过蛋白质印迹法测定的补骨脂素-3-O-葡萄糖醛酸化与UGT1A9蛋白水平之间也观察到强相关性（r = 0.944，p < 0.001）。此外，基于RAF方法,UGT1A9在HLM中负责99.6%的补骨脂素-3-O-葡萄糖醛酸化。总之，补骨脂素可进行有效的葡萄糖醛酸化，根据UGT同工酶生成一种或两种单葡萄糖醛酸苷。此外，补骨脂素-3-O-葡萄糖醛酸化是UGT1A9的一种出色的体外标志物。
