Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
J Pharmacol Exp Ther. 2012 Jan;340(1):46-55. doi: 10.1124/jpet.111.184721. Epub 2011 Oct 4.
Coumadin (R-, S-warfarin) is a challenging drug to accurately dose, both initially and for maintenance, because of its narrow therapeutic range and wide interpatient variability and is typically administered as a racemic (Rac) mixture, which complicates the biotransformation pathways. The goal of the current work was to identify the human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of the separated R- and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin and the possible interactions between these enantiomers. The kinetic and inhibition constants for human recombinant 1A family UGTs toward these separated enantiomers have been assessed using high-performance liquid chromatography (HPLC)-UV-visible analysis, and product confirmations have been made using HPLC-mass spectrometry/mass spectrometry. We found that separated R- and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin demonstrate significantly different glucuronidation kinetics and can be mutually inhibitory. In some cases significant substrate inhibition was observed, as shown by K(m), V(max), and K(i), comparisons. In particular, UGT1A1 and extrahepatic UGT1A10 have significantly higher capacities than other isoforms for S-7-hydroxywarfarin and R-7-hydroxywarfarin glucuronidation, respectively. Activity data generated using a set of well characterized human liver microsomes supported the recombinant enzyme data, suggesting an important (although not exclusive) role for UGT1A1 in glucuronidation of the main warfarin metabolites, including Rac-6- and 7-hydroxywarfarin and their R- and S-enantiomers in the liver. This is the first demonstration that the R- and S-enantiomers of hydroxywarfarins are glucuronidated, with significantly different enzymatic affinity and capacity, and supports the importance of UGT1A1 as the major hepatic isoform involved.
华法林（R-，S-）是一种具有挑战性的药物，无论是初始剂量还是维持剂量，因为它的治疗范围狭窄，个体间差异大，而且通常作为外消旋（Rac）混合物给药，这使得生物转化途径复杂化。目前的工作目标是确定参与 6-、7-和 8-羟基华法林的分离 R-和 S-对映体的葡萄糖醛酸化的人 UDP-葡糖醛酸基转移酶（UGT），以及这些对映体之间可能的相互作用。使用高效液相色谱（HPLC）-紫外可见分析评估了人重组 1A 家族 UGT 对这些分离的对映体的动力学和抑制常数，并使用 HPLC-质谱/质谱进行了产物确认。我们发现，6-、7-和 8-羟基华法林的分离 R-和 S-对映体表现出明显不同的葡萄糖醛酸化动力学，并且可以相互抑制。在某些情况下，如 Km、Vmax 和 Ki 的比较所示，观察到显著的底物抑制。特别是，UGT1A1 和肝外 UGT1A10 对 S-7-羟基华法林和 R-7-羟基华法林的葡萄糖醛酸化具有明显高于其他同工酶的能力。使用一组经过充分表征的人肝微粒体生成的活性数据支持重组酶数据，表明 UGT1A1 在华法林主要代谢物（包括 Rac-6-和 7-羟基华法林及其 R-和 S-对映体）的葡萄糖醛酸化中具有重要作用（尽管不是排他性作用）在肝脏中。这是首次证明羟基华法林的 R-和 S-对映体被葡萄糖醛酸化，具有明显不同的酶亲和力和能力，并支持 UGT1A1 作为主要涉及的肝同工酶的重要性。
