Obtaining Exposures of Metabolites in Preclinical Species through Plasma Pooling and Quantitative NMR: Addressing Metabolites in Safety Testing (MIST) Guidance without Using Radiolabeled Compounds and Chemically Synthesized Metabolite Standards

Abstract

The recent guidance on "Safety Testing of Drug Metabolites" issued by the U.S. Food and Drug Administration, Center for Drug Evaluation and Research (CDER) has highlighted the importance of identifying and characterizing drug metabolites as early as possible in drug discovery and development. Furthermore, upon identifying significant circulating metabolites in human plasma, it has become important to demonstrate that these metabolites are present at an equal or greater exposure level (area under the curve, AUC) in any one of the preclinical species used in safety testing. Frequently, synthetic standards of metabolites are not available, and hence, obtaining their AUC values can be a challenge. In this report, we demonstrate how combinations of nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS), and plasma pooling methods were used to obtain reliable AUC values of metabolites present in the plasma of preclinical species from short-term safety studies. Plasma pooling methods were compared to the traditional approaches of obtaining quantitative information on the levels of circulating metabolites in preclinical species. The exposure values obtained via sample pooling were comparable to those obtained by traditional methods of analyzing samples individually. In the absence of synthetic chemical standards, calculations of AUC values of metabolites, using either sample pooling or traditional approaches, were achieved through the use of UV detectors. In cases where the UV properties of metabolites were significantly different from their parent compounds, NMR was used as a quantitative tool to obtain exposure values. NMR was found to be useful in quantitating biologically produced metabolites, which could subsequently be used as reference compounds for further quantitative studies. The limitations of UV detectors to obtain exposure estimates are discussed. A practical solution is presented that will enable us to obtain a quantitative assessment of metabolite exposure in humans and coverage in toxicology species, hence, circumventing the use of radiolabeled compounds or authentic chemically synthesized standards of metabolites.

Addressing the Challenges of Low Clearance in Drug Research

“…The individual fractions containing metabolites are evaporated, reconstituted in deuterated solvent, and the solution strength is determined using NMR spectroscopy. This procedure has been enabled by the advances in cryomicroprobe technology and magnet strength for NMR such that solutions of less than 50-μL total volume and concentrations of 50 μM can be reliably analyzed (16,17). The resulting solution of metabolite in deuterated solvent can then be used as a mother stock from which dilutions can be made to construct a standard curve for HPLC-MS analysis of rate of metabolite formation in metabolic incubations.…”

Meeting Report: Metabolites in Safety Testing (MIST) Symposium—Safety Assessment of Human Metabolites: What’s REALLY Necessary to Ascertain Exposure Coverage in Safety Tests?

“…The currently available techniques may be integrated to reliably find and structurally identify the metabolites from the plasma and urine samples that are already collected in typical phase I clinical trials. And semiquantitation of metabolites using liquid chromatography (LC)-UV, LC/MS/MS peak area ratio comparison (10,11,13), radiolabeled calibrant (5-7), and quantitative NMR standards (8,9,14) can be employed to compare the exposures to the metabolites in animals to humans. This allows a sponsor to comply with regulatory expectations for metabolite safety assessment without the need to wait for the conventional 14 C-ADME studies that are usually conducted in phases II or III.…”

“…In response to the bioanalytical challenges posed by these expectations, investigators have proactively developed alternate approaches that can offer assurance of relative exposures to metabolites in animals and humans. These include the application of radiometrically calibrated metabolite standards (5-7) or creating metabolite standards from biological sources that are quantitated using nuclear magnetic resonance (NMR) spectroscopy (8,9). Finally, demonstration of relative exposures to metabolites across species can be accomplished using HPLCmass spectrometer (MS) peak area comparisons from animal and human plasma extracts (10,11).…”

A decade of drug metabolite safety testing: industry and regulatory shared learning

“…Some of these approaches include mixed matrix LC-MS/MS peak area comparisons across species [13], NMR-based techniques [14,15], and bioanalytical methods with radiocalibrants [16]. LC-MS peak area comparisons between animals and humans are routinely used by some sponsors to demonstrate that metabolite concentrations in animals exceed plasma concentrations in humans.…”