Optimization of Experimental Conditions of Automated Glucuronidation Assays in Human Liver Microsomes Using a...

UDP-glucuronosyltransferase (UGT)-mediated metabolism is possibly the most important conjugation reaction for marketed drugs. However, there are currently no generally accepted standard incubation conditions for UGT microsomal assays, and substantial differences in experimental design and methodology between laboratories hinder cross-study comparison of in vitro activities. This study aimed to define optimal experimental conditions to determine glucuronidation activity of multiple UGT isoforms simultaneously using human liver microsomes. Hepatic glucuronidation activities of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17 were determined using cocktail incubations of 10 UGT probe substrates. Buffer components and cosubstrates were assessed over a range of concentrations including magnesium chloride (MgCl₂; 0-10 mM) and uridine 5'-diphosphoglucuronic acid (UDPGA; 1-25 mM) with either Tris-HCl or potassium phosphate buffer (100 mM, pH 7.4). Greater microsomal glucuronidation activity by different hepatic UGT isoforms was obtained using 10 mM MgCl₂ and 5 mM UDPGA with 100 mM Tris-HCl buffer. The influence of bovine serum albumin (BSA; 0.1%-2% w/v) on glucuronidation activity was also assessed. Enzyme- and substrate-dependent effects of BSA were observed, resulting in decreased total activity of UGT1A1, UGT1A3, and UGT2B17 and increased total UGT1A9 and UGT2B7 activity. The inclusion of BSA did not significantly reduce the between-subject variability of UGT activity. Future in vitro UGT profiling studies under the proposed optimized experimental conditions would allow high-quality positive control data to be generated across laboratories, with effective control of a high degree of between-donor variability for UGT activity and for chemical optimization toward lower-clearance drug molecules in a pharmaceutical drug discovery setting.