Crystalline Sponges as a Sensitive and Fast Method for Metabolite Identification: Application to Gemfibrozil and its Phase I and II Metabolites

Abstract

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 59-CH 2 OH gemfibrozil (M1), 49-OH gemfibrozil (M2), 59-COOH gemfibrozil (M3), and the acyl glucuronide of gemfibrozil, 1-O-b-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.

Guest-protein incorporation into solvent channels of a protein host crystal (hostal)

“…Sponge systems exploit the small channels of crystalline hosts to accommodate, and optimally to order, small guests, although the fundamental principles of host-guest interactions for the sponge method are not fully understood (Hayes et al, 2016). Recent developments with crystal sponge hosts have allowed the structure determination of a wider range of guests including increasingly larger, more flexible and chemically complex molecules (Yoshioka et al, 2015;Ning et al, 2016;Rosenberger et al, 2020) as well as compounds in aqueous solution (Poel et al, 2019). Commonly used host-guest sponge systems are typically not designed for interactions with a specific guest, and a single crystal host may be used for guests with varying host-guest interactions (Hoshino et al, 2016).…”

“…In this socalled crystal sponge method, guest molecules are incorporated via diffusion into the solvent channels of an existing host crystal, often a metal-organic framework, although metal-free porous crystals have also been used (Inokuma et al, 2013;Sanna et al, 2015;Hoshino et al, 2016). Optimization of this method (Hoshino et al, 2016) has allowed the structure determination of difficult cases, including guest compounds with axial or planar chirality (Yoshioka et al, 2015), small flexible compounds (Ning et al, 2016) and modified metabolites (Rosenberger et al, 2020) or other guest compounds in aqueous solution (Poel et al, 2019). The narrow solvent channels of most organic and metal-organic frameworks limit this technique to small-molecule guests.…”

Metabolic Profiling of S-praziquantel: Structure Elucidation Using the Crystalline Sponge Method in Combination with Mass Spectrometry and Nuclear Magnetic Resonance

“…The supernatants were analyzed and fractionated on an Acquity Arc HPLC system (Waters Corporation). The equipment components were described in detail in our previous work (Rosenberger et al, 2020).…”

“…The incubator temperature was set to 50 °C and the evaporation time was one day. SC-XRD measurements were performed by using a Rigaku Oxford Diffraction XtaLAB Synergy-R diffractometer (Cu-K α , λ = 1.54184 Å) at a temperature of 100 K and crystal structure modeling using OLEX2 (Dolomanov et al, 2009), SHELXT and SHELXL (Sheldrick, 2015) were conducted as described in detail in our previous work (Rosenberger et al, 2020).…”

“…During a "soaking process", the analyte (guest) is absorbed into the pores of the three-dimensional framework via diffusion and regularly ordered by intermolecular, noncovalent interactions and thus accessible for X-ray diffraction analysis (Inokuma et al, 2016;Brunet et al, 2017;Sakurai et al, 2017). We recently demonstrated that the CS-XRD is a valuable tool, which can be applied to identify the exact position of metabolism for phase I and phase II metabolites generated in low amounts from in vitro incubation (Rosenberger et al, 2020).…”