Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Research and Development, Groton, Connecticut
Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Research and Development, Groton, Connecticut.
Drug Metab Dispos. 2024 Sep 16;52(10):1083-1093. doi: 10.1124/dmd.124.001812.
UGT2B4 is a highly expressed drug-metabolizing enzyme in the liver contributing to the glucuronidation of several drugs. To enable quantitatively assessing UGT2B4 contribution toward metabolic clearance, a potent and selective UGT2B4 inhibitor that can be used for reaction phenotyping was sought. Initially, a canagliflozin-2'--glucuronyl transferase activity assay was developed in recombinant UGT2B4 and human liver microsomes (HLM) [±2% bovine serum albumin (BSA)]. Canagliflozin-2'--glucuronidation (C2OG) substrate concentration at half-maximal velocity value in recombinant UGT2B4 and HLM were similar. C2OG formation intrinsic clearance was five- to seven-fold higher in incubations containing 2% BSA, suggesting UGT2B4 susceptibility to the inhibitory unsaturated long-chain fatty acids released during the incubation. Monitoring for C2OG formation, 180 compounds were evaluated for UGT2B4 inhibition potency in the presence and absence of 2% BSA. Compounds that exhibited an apparent UGT2B4 IC of < 1 M in HLM with 2% BSA were evaluated for inhibition of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, UGT2B10, UGT2B15, and UGT2B17 catalytic activities to establish selectivity suitable for supporting UGT reaction phenotyping. In this study, clotrimazole was identified as a potent UGT2B4 inhibitor (HLM apparent IC of 11 to 35 nM ± 2% BSA). Moreover, clotrimazole exhibited selectivity for UGT2B4 inhibition (>24-fold) over the other UGT enzymes evaluated. Additionally, during this study it was discovered that the previously described UGT2B7 inhibitors 16- and 16-phenyllongifolol also inhibit UGT2B4. Clotrimazole, a potent and selective UGT2B4 inhibitor, will prove essential during UGT reaction phenotyping. SIGNIFICANCE STATEMENT: To mechanistically evaluate drug interactions, it is essential to understand the contribution of individual enzymes to the metabolic clearance of a drug. The present study describes the development of a UGT2B4 activity assay that enabled the discovery of the highly selective and potent UGT2B4 inhibitor clotrimazole. Clotrimazole can be used in UGT reaction phenotyping studies to estimate fractional contribution of UGT2B4.
UGT2B4 是肝脏中高度表达的药物代谢酶，有助于几种药物的葡萄糖醛酸化。为了能够定量评估 UGT2B4 对代谢清除率的贡献，需要寻找一种能够用于反应表型分析的强效和选择性 UGT2B4 抑制剂。最初，在重组 UGT2B4 和人肝微粒体 (HLM)[±2%牛血清白蛋白 (BSA)]中开发了一种坎格列净-2'-葡萄糖醛酸转移酶活性测定法。在重组 UGT2B4 和 HLM 中，坎格列净-2'-葡萄糖醛酸化 (C2OG) 底物达到半最大速度值的浓度相似。在含有 2%BSA 的孵育中，C2OG 形成的内在清除率高出五到七倍，这表明 UGT2B4 易受孵育过程中释放的不饱和长链脂肪酸的抑制。在监测 C2OG 形成的过程中，在有和没有 2%BSA 的情况下，评估了 180 种化合物对 UGT2B4 的抑制效力。在 HLM 中，在有 2%BSA 的情况下，对表观 UGT2B4 IC 小于 1μM 的化合物进行了评估，以评估它们对 UGT1A1、UGT1A3、UGT1A4、UGT1A6、UGT1A9、UGT2B7、UGT2B10、UGT2B15 和 UGT2B17 催化活性的抑制作用，以确定适合支持 UGT 反应表型分析的选择性。在这项研究中，克霉唑被确定为一种强效的 UGT2B4 抑制剂（HLM 表观 IC 为 11 至 35 nM±2%BSA）。此外，克霉唑对评估的其他 UGT 酶表现出对 UGT2B4 抑制的选择性（超过 24 倍）。此外，在这项研究中发现，先前描述的 UGT2B7 抑制剂 16-和 16-苯基长叶醇也抑制 UGT2B4。克霉唑是一种强效和选择性的 UGT2B4 抑制剂，在 UGT 反应表型分析中证明是必不可少的。意义声明：为了从机制上评估药物相互作用，了解单个酶对药物代谢清除率的贡献至关重要。本研究描述了 UGT2B4 活性测定法的开发，该测定法使发现高度选择性和强效的 UGT2B4 抑制剂克霉唑成为可能。克霉唑可用于 UGT 反应表型研究中，以估计 UGT2B4 的分数贡献。
