Determination of Degradation Pathways and Kinetics of Acyl Glucuronides by NMR Spectroscopy

Abstract

Acyl glucuronides have been implicated in the toxicity of many xenobiotics and marketed drugs. These toxicities are hypothesized to be a consequence of covalent binding of the reactive forms of the acyl glucuronide to proteins. Reactive intermediates of the acyl glucuronide arise from the migration of the aglycone leading to other positional and stereoisomers under physiological conditions. In order to screen for the potential liabilities of these metabolites during the early phase of pharmaceutical development, an NMR method based on the disappearance of the anomeric resonance of the O-1-acyl glucuronide was used to monitor the degradation kinetics of 11 structurally diverse acyl glucuronides, including those produced from the known nonsteroidal anti-inflammatory drugs (NSAIDs). The acyl glucuronides were either chemically synthesized or were isolated from biological matrices (bile, urine, and liver microsomal extracts). The half-lives attained utilizing this method were found to be comparable to those reported in the literature. NMR analysis also enabled the delineation of the two possible pathways of degradation: acyl migration and hydrolytic cleavage. The previously characterized 1H resonances of acyl migrated products are quite distinguishable from those that arise from hydrolysis. The NMR method described here could be used to rank order acyl glucuronide forming discovery compounds based on the potential reactivity of the conjugates and their routes of decomposition under physiological conditions. Furthermore, we have shown that in vitro systems such as liver microsomal preparations can be used to generate sufficient quantities of acyl glucuronides from early discovery compounds for NMR characterization. This is particularly important, as we often have limited supply of early discovery compounds to conduct in vivo studies to generate sufficient quantities of acyl glucuronides for further characterization.

Metabolic Activation of Mefenamic Acid Leading to Mefenamyl-S-Acyl-Glutathione Adduct Formation In Vitro and In Vivo in Rat

“…The 1.3-and 6-fold GSTcatalyzed increase in MFA-SG formation rates from reactions of MFA-SCoA and MFA-1-␤-O-G with GSH is consistent with the weak catalysis observed in previous studies with clofibric acid derivatives, where rat liver GST-mediated catalysis was shown to occur in incubations of clofibryl-1-␤-O-G (ϳ8-fold; Shore et al, 1995) and clofibryl-S-acyl-CoA (ϳ3-fold; Grillo and Benet, 2002) with GSH forming clofibryl-S-acyl-GSH thioester. In addition, we determined MFA-SCoA to be chemically stable in buffer at pH 7.4 and 37°C over 15 h of incubation, whereas MFA-1-␤-O-G degraded with a t 1/2 of 16.5 h, which was the same as reported previously (McGurk et al, 1996;Walker et al, 2007). The degradation of MFA-1-␤-O-G in buffer is known to occur almost exclusively through acyl migration and not by hydrolysis (Walker et al, 2007).…”

Stereoselective Inhibition of Methotrexate Excretion by Glucuronides of Nonsteroidal Anti-inflammatory Drugs via Multidrug Resistance Proteins 2 and 4

“…Since reaction mixture was incubated for 20 minutes in the MRP2-mediated transport experiment, part of the 1-Ob-glucuronides probably decomposed. Based on the published degradation rate constants or elimination half-lives of glucuronides at pH 7.4 (Iwaki et al, 1998;Walker et al, 2007;Sawamura et al, 2010), the remaining unchanged 1-Oglucuronides are calculated to be 68%-72% for diclofenac glucuronide, 81% for R-naproxen glucuronide, 89%-93% for S-naproxen glucuronide, and 92% for rac-ibuprofen glucuronide after 20-minute incubation at pH 7.4. We re-evaluated the stability of the glucuronides in the reaction buffer used in a MRP-mediated transport experiment.…”

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

“…Fraction A (500 ml) was stopped by adding 500 ml of ice-cold acetonitrile containing 10% (v/v) of acetic acid, as acyl migration may be stabilized under an acidic condition (pH , 4.0) according to previous publications (Walker et al, 2007;Di Meo et al, 2013). The sample was mixed thoroughly and then centrifuged at 16,000g at 4°C for 10 minutes.…”

“…Therefore the application of the acyl migration method to assess reactivity of those compounds for ranking and selection may provide misestimated results. However, the acyl migration method was still applicable to a majority of drugs that have acyl glucuronide issues, as it has been reported that acyl migration was identified as the predominant reaction occurring in vitro (Walker et al, 2007). More importantly, the acyl migration method can provide preliminary reactivity information in a rapid screening assay that can be used to support the selection of low-risk drug candidates in the drug discovery phase that do not depend on authentic standards.…”