Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut.
Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut
Drug Metab Dispos. 2020 Aug;48(8):645-654. doi: 10.1124/dmd.120.090696. Epub 2020 May 30.
Human liver microsomes (HLM) are a commonly used tool to study drug metabolism in vitro. Typical experiments conducted using suspensions of HLM can be challenging to separate from the incubation solution without lengthy ultracentrifugation steps. Magnetizable beads coated with silica (MGBS) were found to bind strongly to HLM, which could then be isolated and purified using a magnet. Binding of HLM to the MGBS (HLM-MGBS) was demonstrated to be mediated by strong interactions between microsomal phospholipids and MGBS, as artificially prepared phosphatidylcholine (PC) liposomes could be more efficiently captured by the MGBS. HLM-MGBS complexes retained functional cytochrome P450 and uridine-diphosphate-glucuronosyltransferase (UGT) activity as indicated by CYP2C8-mediated amodiaquine de-ethylation, CYP3A4-mediated midazolam 1'hydroxylation, UGT1A1-mediated glucuronidation of estradiol, UGT1A9-mediated glucuronidation of propofol, and UGT2B7-mediated glucuronidation of zidovudine. When comparing suspension HLM alone with HLM-MGBS complexes containing equivalent amounts of HLM, the intrinsic clearance (CL) of CYP450 substrates was comparable; however, CL of UGT1A1, UGT1A9, and UGT2B7 was increased in the HLM-MGBS system between 1.5- and 6-fold. HLM-MGBS used in an incubation could also be readily replaced with fresh HLM-MGBS to maintain the presence of active enzymes. Thus, HLM-MGBS demonstrate increased in vitro metabolic efficiency and manipulability, providing a new platform for determination of accurate metabolic parameters. SIGNIFICANCE STATEMENT: The following work describes the strong binding of HLM to magnetizable beads. In addition, the preservation of enzyme activity on the bound HLM provides a novel means to conduct preclinical metabolism studies.
人肝微粒体（HLM）是体外研究药物代谢的常用工具。使用 HLM 悬浮液进行的典型实验在没有冗长的超速离心步骤的情况下，难以与孵育溶液分离。研究发现，涂有二氧化硅的可磁化珠（MGBS）与 HLM 结合紧密，然后可以使用磁铁将其分离和纯化。HLM 与 MGBS 的结合（HLM-MGBS）被证明是由微粒体磷脂和 MGBS 之间的强相互作用介导的，因为人工制备的磷脂酰胆碱（PC）脂质体可以更有效地被 MGBS 捕获。HLM-MGBS 复合物保留了功能性细胞色素 P450 和尿苷二磷酸-葡萄糖醛酸转移酶（UGT）活性，如 CYP2C8 介导的氨苯砜去乙基化、CYP3A4 介导的咪达唑仑 1'羟化、UGT1A1 介导的雌二醇葡萄糖醛酸化、UGT1A9 介导的丙泊酚葡萄糖醛酸化和 UGT2B7 介导的齐多夫定葡萄糖醛酸化。当将单独的悬浮 HLM 与含有等量 HLM 的 HLM-MGBS 复合物进行比较时，CYP450 底物的内在清除率（CL）相当；然而，在 HLM-MGBS 系统中，UGT1A1、UGT1A9 和 UGT2B7 的 CL 增加了 1.5-6 倍。孵育中使用的 HLM-MGBS 也可以很容易地用新鲜的 HLM-MGBS 代替，以保持活性酶的存在。因此，HLM-MGBS 表现出更高的体外代谢效率和可操作性，为确定准确的代谢参数提供了一个新的平台。意义陈述：这项工作描述了 HLM 与可磁化珠的强结合。此外，结合的 HLM 上酶活性的保留提供了一种新的方法来进行临床前代谢研究。
