Glycerolysis of Acyl Glucuronides as an Artifact of in Vitro Drug Metabolism Incubations

Abstract

ABSTRACT:During an investigation of the in vitro glucuronidation of benoxaprofen by human liver S-9 fraction, an unusual drug-related entity possessing a protonated molecular ion that was 74 mass units greater than the parent drug was observed. It was identified as the glycerol ester of benoxaprofen. Formation of this entity required inclusion of uridine diphosphoglucuronic acid (UDPGA) in the incubation, suggesting the formation of benoxaprofen acyl glucuronide followed by transesterification with the glycerol present in the incubation due to its presence as a stabilizer for liver subcellular fractions. Formation occurred during the sample work-up procedure while the samples were subjected to evaporation in vacuo, which does not remove glycerol. Conversion of purified benoxaprofen acyl glucuronide to the glycerol ester was demonstrated in glycerol at 37°C. Other drugs that are converted to acyl glucuronides in vitro (diclofenac, mefenamic acid, tolmetin, and naproxen) were also shown to form corresponding glycerol esters when incubated with human liver S-9 fraction and UDPGA. The potential formation of glycerol esters of carboxylic acid drugs undergoing acyl glucuronidation in vitro represents an experimental artifact to which drug metabolism scientists should be aware.

High-Resolution Mass Spectrometry Elucidates Metabonate (False Metabolite) Formation from Alkylamine Drugs during In Vitro Metabolite Profiling

“…Artifact formation from acyl glucuronides as a direct result of the presence of glycerol in human liver S9 fraction has been reported (Obach, 2009). However, the generation of aldehydes as byproducts of N-dealkylation reactions in the absence of glycerol or methanol is unavoidable; in the case of methylamines, N-demethylation will necessarily produce formaldehyde.…”

Discovery of N‐Aryloxypropylbenzylamines as Voltage‐Gated Sodium Channel NaV1.2‐Subtype‐Selective Inhibitors

“…Hepatic blood clearance and the corresponding hepatic extraction ratio (E H )w ere calculated using the well-stirred model of hepatic extraction in each species, according to the "in vitro T 1/2 "a pproach described in Obach. [31] The E H was then used to classify compounds as low (< 0.3), intermediate (0.3-0.7), high (0.7-0.95) or very high (> 0.95) extraction compounds. Predicted in vivo clearance values have not been corrected for microsomal or plasma protein binding.…”

Synthesis, QSAR studies, and metabolic stability of novel 2‐alkylthio‐4‐chloro‐N‐(5‐oxo‐4,5‐dihydro‐1,2,4‐triazin‐3‐yl)benzenesulfonamide derivatives as potential anticancer and apoptosis‐inducing agents

“…Metabolic half-life (t 1/2 ) was calculated from the slope of the linear regression. [20] LC-MS analysis was performed on an Agilent 1260 system coupled to SingleQuad 6120 mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Ascentis Express ES-CN (2.1 mm × 100 mm, please confirm 2.7 μm, Supelco Analytical, Bellefonte, PA, USA) was used in reversed-phase mode with gradient elution starting with 50% of phase A (10 mm ammonium formate in water) and 50% of phase B (10 mm ammonium formate in acetonitrile-water mixture, 95:5 v/v).…”