RT Journal Article
SR Electronic
T1 Crystalline Sponges as a Sensitive and Fast Method for Metabolite Identification: Application to Gemfibrozil and its Phase I and II Metabolites
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 587
OP 593
DO 10.1124/dmd.120.091140
VO 48
IS 7
A1 Lara Rosenberger
A1 Carolina von Essen
A1 Anupam Khutia
A1 Clemens Kühn
A1 Klaus Urbahns
A1 Katrin Georgi
A1 Rolf W. Hartmann
A1 Lassina Badolo
YR 2020
UL http://dmd.aspetjournals.org/content/48/7/587.abstract
AB 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′-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 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.