Discovery and Development Technologies (DDTech) (L.R., K.U., K.G., L.B.) and Innovation Center (C.v.E., A.K., C.K.), Merck KGaA, Darmstadt, Germany; and Department of Drug Design and Optimization (DDOP), Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS)-Helmholtz Centre for Infection Research (HZI), Saarbrücken, Germany (L.R., R.W.H.).
Discovery and Development Technologies (DDTech) (L.R., K.U., K.G., L.B.) and Innovation Center (C.v.E., A.K., C.K.), Merck KGaA, Darmstadt, Germany; and Department of Drug Design and Optimization (DDOP), Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS)-Helmholtz Centre for Infection Research (HZI), Saarbrücken, Germany (L.R., R.W.H.)
Drug Metab Dispos. 2020 Jul;48(7):587-593. doi: 10.1124/dmd.120.091140. Epub 2020 May 20.
Understanding the metabolism of new drug candidates is important during drug discovery and development, as circulating metabolites may contribute to efficacy or cause safety issues. In the early phase of drug discovery, human in vitro systems are used to investigate human relevant metabolism. Though conventional techniques are limited in their ability to provide complete molecular structures of metabolites (liquid chromatography mass spectrometry) or require a larger amount of material not available from in vitro incubation (nuclear magnetic resonance), we here report for the first time the use of the crystalline sponge method to identify phase I and phase II metabolites generated from in vitro liver microsomes or S9 fractions. Gemfibrozil was used as a test compound. Metabolites generated from incubation with microsomes or S9 fractions, were fractionated using online fraction collection. After chromatographic purification and fractionation of the generated metabolites, single crystal X-ray diffraction of crystalline sponges was used to identify the structure of gemfibrozil metabolites. This technique allowed for complete structure elucidation of 5'-CHOH gemfibrozil (M1), 4'-OH gemfibrozil (M2), 5'-COOH gemfibrozil (M3), and the acyl glucuronide of gemfibrozil, 1-O--glucuronide (M4), the first acyl glucuronide available in the Cambridge Crystallographic Data Centre. Our study shows that when optimal soaking is possible, crystalline sponges technology is a sensitive (nanogram amount) and fast (few days) method that can be applied early in drug discovery to identify the structure of pure metabolites from in vitro incubations. SIGNIFICANCE STATEMENT: Complete structure elucidation of human metabolites plays a critical role in early drug discovery. Low amounts of material (nanogram) are only available at this stage and insufficient for nuclear magnetic resonance analysis. The crystalline sponge method has the potential to close this gap, as demonstrated in this study.
理解新候选药物的代谢对于药物发现和开发非常重要，因为循环代谢物可能会影响疗效或导致安全性问题。在药物发现的早期阶段，人们使用人体体外系统来研究与人体相关的代谢。尽管传统技术在提供代谢物的完整分子结构方面能力有限（液相色谱-质谱联用），或者需要更多的无法从体外孵育获得的材料（核磁共振），但我们首次报道了使用晶体海绵方法来鉴定来自体外肝微粒体或 S9 级分的 I 相和 II 相代谢物。吉非罗齐被用作测试化合物。使用在线馏分收集法对与微粒体或 S9 级分孵育生成的代谢物进行分级。在生成的代谢物经过色谱纯化和分级后，使用晶体海绵的单晶 X 射线衍射来鉴定吉非罗齐代谢物的结构。该技术可实现 5'-CHOH 吉非罗齐（M1）、4'-OH 吉非罗齐（M2）、5'-COOH 吉非罗齐（M3）和吉非罗齐的酰基葡萄糖醛酸 1-O--葡萄糖醛酸苷（M4）的完全结构解析，这是首个在剑桥晶体数据中心可用的酰基葡萄糖醛酸苷。我们的研究表明，当最佳浸泡成为可能时，晶体海绵技术是一种灵敏（纳克级）且快速（数天）的方法，可以在药物发现的早期应用于从体外孵育中鉴定纯代谢物的结构。 意义：人类代谢物的完全结构解析在早期药物发现中起着关键作用。在这个阶段，只有少量的材料（纳克级）可用，且不足以进行核磁共振分析。晶体海绵方法有潜力弥补这一差距，正如本研究所示。
