Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Inc., Groton, Connecticut 06340, USA.
Drug Metab Dispos. 2012 May;40(5):1051-65. doi: 10.1124/dmd.111.043117. Epub 2012 Feb 22.
The measurement of the effect of new chemical entities on human UDP-glucuronosyltransferase (UGT) marker activities using in vitro experimentation represents an important experimental approach in drug development to guide clinical drug-interaction study designs or support claims that no in vivo interaction will occur. Selective high-performance liquid chromatography-tandem mass spectrometry functional assays of authentic glucuronides for five major hepatic UGT probe substrates were developed: β-estradiol-3-glucuronide (UGT1A1), trifluoperazine-N-glucuronide (UGT1A4), 5-hydroxytryptophol-O-glucuronide (UGT1A6), propofol-O-glucuronide (UGT1A9), and zidovudine-5'-glucuronide (UGT2B7). High analytical sensitivity permitted characterization of enzyme kinetic parameters at low human liver microsomal and recombinant UGT protein concentration (0.025 mg/ml), which led to a new recommended optimal universal alamethicin activation concentration of 10 μg/ml for microsomes. Alamethicin was not required for recombinant UGT incubations. Apparent enzyme kinetic parameters, particularly for UGT1A1 and UGT1A4, were affected by nonspecific binding. Unbound intrinsic clearance for UGT1A9 and UGT2B7 increased significantly after addition of 2% bovine serum albumin, with minimal changes for UGT1A1, UGT1A4, and UGT1A6. Eleven potential UGT and cytochrome P450 inhibitors were evaluated as UGT inhibitors, resulting in observation of nonselective UGT inhibition by chrysin, mefenamic acid, silibinin, tangeretin, ketoconazole, itraconazole, ritonavir, and verapamil. The pan-cytochrome P450 inhibitor, 1-aminobenzotriazole, minimally inhibited UGT activities and may be useful in reaction phenotyping of mixed UGT and cytochrome P450 substrates. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of human UGT activities and the identification of UGT enzyme-selective chemical inhibitors.
使用体外实验测量新化学实体对人 UDP-葡萄糖醛酸基转移酶 (UGT) 标记物活性的影响，是药物开发中一种重要的实验方法，可用于指导临床药物相互作用研究设计或支持无体内相互作用的结论。针对五种主要肝 UGT 探针底物的真实葡萄糖醛酸苷的选择性高效液相色谱-串联质谱功能测定法得到了开发：β-雌二醇-3-葡萄糖醛酸苷 (UGT1A1)、三氟拉嗪-N-葡萄糖醛酸苷 (UGT1A4)、5-羟基色氨酸-O-葡萄糖醛酸苷 (UGT1A6)、丙泊酚-O-葡萄糖醛酸苷 (UGT1A9) 和齐多夫定-5'-葡萄糖醛酸苷 (UGT2B7)。高分析灵敏度使得能够在人肝微粒体和重组 UGT 蛋白浓度较低的情况下 (0.025 mg/ml) 对酶动力学参数进行表征，这导致了新的推荐通用拉米夫定激活浓度为 10 μg/ml 用于微粒体。重组 UGT 孵育不需要拉米夫定。非特异性结合会影响表观酶动力学参数，特别是 UGT1A1 和 UGT1A4。添加 2%牛血清白蛋白后，UGT1A9 和 UGT2B7 的未结合内在清除率显著增加，而 UGT1A1、UGT1A4 和 UGT1A6 的变化很小。评价了 11 种潜在的 UGT 和细胞色素 P450 抑制剂作为 UGT 抑制剂，结果观察到白杨素、甲芬那酸、水飞蓟素、橙皮苷、酮康唑、伊曲康唑、利托那韦和维拉帕米对 UGT 具有非选择性抑制作用。泛细胞色素 P450 抑制剂 1-氨基苯并三唑对 UGT 活性的抑制作用最小，可能有助于混合 UGT 和细胞色素 P450 底物的反应表型鉴定。这些方法应可用于常规评估新候选药物通过抑制人 UGT 活性引起药代动力学药物相互作用的潜力，并鉴定 UGT 酶选择性化学抑制剂。
