Abstract

An in vitro screening model was developed to determine the reactivity of acyl glucuronide metabolites from carboxylic drugs. This assay is composed of two phases. The first is a phase of biosynthesis of acyl glucuronides by human liver microsomes (HLM). The second, during which acyl glucuronides are incubated with human serum albumin (HSA), consists of assessing the reactivity of acyl glucuronides toward HSA. Both phases are performed successively in the same experiment. This model was validated using eight carboxylic drugs that were well known for their reactivity, their extent of covalent binding, and their immunological potential. These products were representative of the scale of reactivity. Each compound was incubated with HLM at 400 μM and metabolized into acyl glucuronide to different extents, ranging from 5.6% (tolmetin) to 89.4% (diclofenac). The first-order aglycone appearance rate constant and the extent of covalent binding to proteins were assayed during the incubation of acyl glucuronides formed with HSA for 24 h. Extensive isomerization phenomenon was observed for each acyl glucuronide between the two phases. An excellent correlation was observed (r², 0.94) between the extent of drug covalent binding to albumin and the aglycone appearance constant weighted by the percentage of isomerization. This correlation represents an in vitro reactivity scale, which will be helpful in drug discovery support programs to predict the covalent binding potential of new chemical entities. This screening model will also allow the comparison of acyl glucuronide reactivity for related structure compounds.