A New Rapid In Vitro Assay for Assessing Reactivity of Acyl Glucuronides

Abstract

Idiosyncratic drug toxicity is a major challenge for the pharmaceutical industry since complex and multifactorial steps are involved, the dose-dependency is unclear, and its occurrence is not reliably predictable. Whereas the exact mechanisms leading to idiosyncratic toxicity remain elusive in many cases, there are often hints at the involvement of reactive metabolites, such as acyl glucuronides formed by conjugation of carboxylic acids with glucuronic acid. Because the patient-related susceptibilities leading to idiosyncratic toxicity are not sufficiently understood, the best option for the pharmaceutical industry is to minimize drug-related risk factors such as potential acyl glucuronide formation. Here, we describe a rapid in vitro assay for the assessment of the reactivity of acyl glucuronides, on the basis of acyl glucuronide migration, that can support the selection of low-risk drug candidates in the drug discovery phase. Twenty marketed compounds with a wide range of half-lives were tested, their acyl glucuronide migration rates were determined and compared with the half-lives of the respective acyl glucuronides. Ranking of acyl glucuronide stability using this method compared well with the results from existing methodologies. With this method, migration rates >20% would indicate higher risk of reactivity. This simpler approach using the acyl glucuronide migration rate is not dependent on authentic standards, therefore eliminating the requirement for either lengthy chemical synthesis or in vitro biosynthesis and purification of the 1-O-b-glucuronide. This methodology provides a rapid in vitro assay to assess acyl glucuronide stability and reactivity that is well suited for use early in the drug discovery phase.

New Perspectives on Acyl Glucuronide Risk Assessment in Drug Discovery: Investigation of In vitro Stability, In situ Reactivity, and Bioactivation

“…This is useful in case the compounds of interest have multiple groups which may undergo conjugation reactions. Stability assays, either via biosynthesis or in rare cases where reference standards of glucuronides are available, set the stage for inferring half-lives [22, 23] and number of peaks in LC-MS n analysis as shown in the present investigation (Supplemental Fig. 2 ).…”

“…These reports however typically only focus on one aspect, often metabolite stability differs for each category [19] or in vitro or in vivo metabolite conjugation of a withdrawn drug [20, 21]. The stability of acyl glucuronides has been studied in buffers, plasma, or human serum albumin [22, 23]. Additionally, reactivity with peptides [24] or evaluation of the disappearance of the anomeric resonance of 1-β-acyl glucuronide by NMR [25] are acknowledged as reasonable surrogates for biochemical reactivity.…”

Metabolic profiling of new synthetic cannabinoids AMB and 5F‐AMB by human hepatocyte and liver microsome incubations and high‐resolution mass spectrometry

“…(H)). Acyl glucuronides tend to elute as clusters, suggesting three acyl glucuronide metabolites that isomerize by intramolecular acyl migration . The glucuronidated metabolite A8 generated from A17 shared fragments m / z 161.0340 and 231.1124 with A17.…”

“…Acyl glucuronides tend to elute as clusters, suggesting three acyl glucuronide metabolites that isomerize by intramolecular acyl migration. [34,35] The glucuronidated metabolite A8 generated from A17 shared fragments m/z 161.0340 and 231.1124 with A17. The position of the glucuronic acid was determined as the indazole core by fragments m/z 161.0340, 407.1448 and 478.2176 ( Fig.…”

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

“…Acyl glucuronides especially play an important role in the assessment of drug-related risk factors, as they contain the risk to be potentially reactive metabolites. An intramolecular rearrangement process, so called "migration," leads to the formation of chemically reactive species that can covalently bind proteins (Bailey and Dickinson, 2003;Zhong et al, 2015).…”