Crystalline Sponges as a sensitive and fast method for metabolite identification: Application to gemfibrozil and its phase I and II metabolites.

: 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 early phase of drug discovery, human in vitro systems are used to investigate human relevant metabolism. While conventional techniques are limited in their ability to provide complete molecular structures of metabolites (liquid chromatography mass spectrometry (LC-MS)) or require larger amount of material not available from in vitro incubation (nuclear magnetic resonance (NMR)), we here report for the first time the use of the crystalline sponge method (CS-XRD) 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 (SC-XRD) of crystalline sponges (CS) was used to identify the structure of gemfibrozil metabolites. This technique allowed for complete structure elucidation of 5'-CH2OH-gemfibrozil (M1), 4'-OH gemfibrozil (M2), 5'-COOH gemfibrozil (M3) and the acyl glucuronide of gemfibrozil, 1-O-β-glucuronide (M4), the first acyl glucuronide available in the Cambridge Crystallographic Data Centre. Our study shows that crystalline sponges technology is a sensitive (ng amount) and fast (few days) method that can be applied early in drug discovery to identify the structure of 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 (ng) are only available at this stage and insufficient for NMR analysis. The crystalline sponge method has the potential to close this gap, as it allows complete structure elucidation of drug metabolites within few days from in vitro incubations.