A Simple Liquid Chromatography-Tandem Mass Spectrometry Method to Determine Relative Plasma Exposures of Drug Metabolites across Species for Metabolite Safety Assessments (Metabolites in Safety Testing). II. Application to Unstable Metabolites

Abstract

ABSTRACT:We previously described a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine relative plasma exposures of drug metabolites across species for metabolite safety assessments. It offers time-and resource-sparing advantages to ascertain metabolite exposure comparisons between humans and laboratory animal species for stable metabolites with high confidence. In this study, we tested the limitation of the methodology with compounds possessing six substituents found in unstable metabolites. Stabilization procedures were used, and stabilized samples were compared with untreated samples for structures with established stabilization processes. In most cases, the parent compounds with established stability were used as the intrinsic stability references except in cases in which the metabolite was more stable than the parent compound. Long-term storage stability of the unstable structures was tested by comparing the response ratio of the metabolite to the stability reference compound for multiple independent analyses covering the storage duration. Autosampler stability was tested using the same response ratio of the reinjections of the reconstituted solution overnight over the first injections. The results supported that the possibility that an abbreviated LC-MS/MS peak area ratio comparison can be applied to epoxide, amide, catechol, and acyl glucuronides to determine the relative plasma exposure of drug metabolites across species; but it may not be suitable for iminium ions and esters. Stability of suspected unstable metabolites can be tested using the methodology described above.

Pragmatic Approaches to Determine the Exposures of Drug Metabolites in Preclinical and Clinical Subjects in the MIST Evaluation of the Clinical Development Phase

“…Regardless of compound, the overall MIST evaluation has to reach a point where the project can ensure that metabolite levels formed in humans have been safety tested prior to efficacy testing in larger human populations. Different ways of quantifying circulating metabolites have been described, e.g., radioactivity, response factors, validated bioanalytical assays, and relative methods, all involving varying degrees of complexity, effort, and cost. − ,− ,, Although the ICH MIST guideline does not specify that metabolites should be monitored at steady-state exposure, it is our view that this is desirable to cover potentially accumulating metabolites. Since the only truly quantitative metabolite profiling studies in preclinical species and humans are the radiolabeled single dose studies, data from all such studies needs to be combined.…”

“…Since the guidelines on MIST have been made available, there has been much discussion in the literature about how to fulfill these expectations from choice of bioanalytical methodology ,− to an overall flow-chart of where these analyses fit into the scheme of drug discovery and clinical development. ,,− ,, AstraZeneca, like other pharmaceutical companies, established an internal strategy for the safety assessment of drug metabolites that aims to satisfy both good science as well as the ICH M3 (R2) guidelines described above and which has been presented in part by Isin et al In our experience, the MIST strategy is only one of many strategies taken into consideration during the planning of a drug development program and most often is not the defining strategy. While there are many published articles of MIST strategies and theoretical reasoning demonstrating the possible impact of the new guidelines, there are to our knowledge few real life, published reports of prospective applications of the various companies’ strategies and the lessons learned from them. ,− , Through the presentation of cases, the present article describes how the AstraZeneca Neuroscience ADME group deepened its understanding of the measurement of metabolite exposure and thereby developed its MIST strategy postguidelines. The actions described in the cases reflect an evolving MIST strategy, in that we were focused on trying to fulfill the regulatory guideline of the day (FDA or ICH) in an ongoing development project with the biological samples at hand.…”

“…While there are many published articles of MIST strategies and theoretical reasoning demonstrating the possible impact of the new guidelines, there are to our knowledge few real life, published reports of prospective applications of the various companies' strategies and the lessons learned from them. 9,[25][26][27][28][29][30][31][32]35 Through the presentation of cases, the present article describes how the AstraZeneca Neuroscience ADME group deepened its understanding of the measurement of metabolite exposure and thereby developed its MIST strategy postguidelines. The actions described in the cases reflect an evolving MIST strategy, in that we were focused on trying to fulfill the regulatory guideline of the day (FDA or ICH) in an ongoing development project with the biological samples at hand.…”

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

“…A simple HPLC-MS/MS method was presented whereby quantitative comparisons of exposures to metabolites between animal and human can be obtained in the absence of authentic standards of the metabolites, calibration curves, and other attributes of standard bioanalytical methods (10,13). A statistical analysis showed that if the experimentally determined, animal-to-human MS response ratio is ≥2.0 then the actual exposure ratio is unity or greater (p<0.01).…”

“…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.…”