Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, Washington (A.M.T., M.G.M., J.P.K., O.T.P., M.S., D.W., A.E.R.); Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich-Heine University, Düsseldorf, Germany (K.R., H.H., C.W.); and Department of Pediatrics III, University, Children's Hospital Essen, University of Duisburg-Essen, Essen, Germany (H.H.)
Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, Washington (A.M.T., M.G.M., J.P.K., O.T.P., M.S., D.W., A.E.R.); Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich-Heine University, Düsseldorf, Germany (K.R., H.H., C.W.); and Department of Pediatrics III, University, Children's Hospital Essen, University of Duisburg-Essen, Essen, Germany (H.H.).
J Pharmacol Exp Ther. 2019 Feb;368(2):308-316. doi: 10.1124/jpet.118.249771. Epub 2018 Nov 8.
A potential CYP4B1 suicide gene application in engineered T-cell treatment of blood cancers has revived interest in the use of 4-ipomeanol (IPO) in gene-directed enzyme prodrug therapy, in which disposition of the administered compound may be critical. IPO contains one chiral center at the carbon bearing a secondary alcohol group; it was of interest to determine the effect of stereochemistry on 1) CYP4B1-mediated bioactivation and 2) (UGT)-mediated glucuronidation. First, ()-IPO and ()-IPO were synthesized and used to assess cytotoxicity in HepG2 cells expressing rabbit CYP4B1 and re-engineered human CYP4B1, where the enantiomers were found to be equipotent. Next, a sensitive UPLC-MS/MS assay was developed to measure the IPO-glucuronide diastereomers and product stereoselectivity in human tissue microsomes. Human liver and kidney microsomes generated () and ()-IPO-glucuronide diastereomers in ratios of 57:43 and 79:21, respectively. In a panel of 13 recombinantly expressed UGTs, UGT1A9 and UGT2B7 were the major isoforms responsible for IPO glucuronidation. ()-IPO-glucuronide diastereoselectivity was apparent with each recombinant UGT, except UGT2B15 and UGT2B17, which favored the formation of ()-IPO-glucuronide. Incubations with IPO and the UGT1A9-specific chemical inhibitor niflumic acid significantly decreased glucuronidation in human kidney, but only marginally in human liver microsomes, consistent with known tissue expression patterns of UGTs. We conclude that IPO glucuronidation in human kidney is mediated by UGT1A9 and UGT2B7. In human liver, it is mediated primarily by UGT2B7 and, to a lesser extent, UGT1A9 and UGT2B15. Overall, the lack of pronounced stereoselectivity for IPO's bioactivation in CYP4B1-transfected HepG2 cells, or for hepatic glucuronidation, suggests the racemate is an appropriate choice for use in suicide gene therapies.
一种潜在的 CYP4B1 自杀基因在工程 T 细胞治疗血液癌症中的应用，重新引起了人们对 4-异丙烯基吗啡（IPO）在基因定向酶前体药物治疗中的应用的兴趣，在这种治疗中，给予化合物的处置可能是关键。IPO 在带有仲醇基团的碳原子上含有一个手性中心；确定立体化学对 1）CYP4B1 介导的生物激活和 2）（UGT）介导的葡萄糖醛酸化的影响很有意义。首先，（）-IPO 和（）-IPO 被合成并用于评估在表达兔 CYP4B1 和重新设计的人 CYP4B1 的 HepG2 细胞中的细胞毒性，其中发现对映异构体具有同等效力。接下来，开发了一种灵敏的 UPLC-MS/MS 测定法来测量人组织微粒体中的 IPO-葡萄糖醛酸化物非对映异构体和产物立体选择性。人肝和肾微粒体分别以 57:43 和 79:21 的比例生成（）和（）-IPO-葡萄糖醛酸化物非对映异构体。在 13 种重组表达的 UGT 中，UGT1A9 和 UGT2B7 是负责 IPO 葡萄糖醛酸化的主要同工酶。（）-IPO-葡萄糖醛酸化物非对映异构体选择性在每种重组 UGT 中均明显，除 UGT2B15 和 UGT2B17 外，它们有利于（）-IPO-葡萄糖醛酸化物的形成。用 IPO 和 UGT1A9 特异性化学抑制剂 niflumic 酸孵育显著降低了人肾中的葡萄糖醛酸化，但在人肝微粒体中仅略有降低，与 UGT 的已知组织表达模式一致。我们得出结论，人肾中的 IPO 葡萄糖醛酸化由 UGT1A9 和 UGT2B7 介导。在人肝中，主要由 UGT2B7 介导，其次是 UGT1A9 和 UGT2B15。总的来说，CYP4B1 转染的 HepG2 细胞中 IPO 的生物激活或肝葡萄糖醛酸化缺乏明显的立体选择性，表明外消旋体是自杀基因治疗的合适选择。
