P25 INDUSTRY STANDARD UGT1A3 PROBE SUBSTRATE CDCA: A SECOND LOOK

Jonathan J. Novak , Pfizer, Inc., Groton, CT
Jian Lin , Pharmacokinetics, Dynamics, and Metabolism, Pfizer, Inc., Groton, CT
Keith Riccardi , Pdm, Pfizer, Inc., Groton, CT
Theunis C. Goosen , Pdm, Pfizer Inc., Groton, CT
Kimberly Lapham , Pharmacokinetics, Dynamics and Metabolism, Pfizer, Inc., Groton, CT
The use of prescription medicines by all age groups has been progressively on the rise throughout the past decade, with the average number of prescribed drugs per patient also increasing. This has led to a need for assessing potential drug-drug interactions (DDIs) early within the drug development process. The phase II conjugating enzyme family UDP-glucuronosyltransferases (UGTs), particularly major hepatic isoforms -1A1, -1A3, -1A4, -1A6, -1A9, -2B7, and -2B15, are of recent interest due to their increasing role in the metabolism and clearance of drugs and xenobiotics. A number of selective probe substrates already exist for many of these isoforms and have been optimized to assess inhibition potential in a human liver microsome (HLM) system1. In 2006, it was reported that UGT1A3 is the major contributing enzyme to chenodeoxycholic acid (CDCA) acyl-glucuronidation in the liver, resulting in the major glucuronide metabolite CDCA-24G2. As such, CDCA was adopted by numerous pharmaceutical companies and vendors as a probe substrate for in vitro UGT1A3 inhibition assays. The objective of this investigation is to assess the selectivity of CDCA toward UGT1A3 with the intent of developing a UGT1A3 enzyme activity assay. CDCA-24G formation kinetics was assessed in pooled HLMs (N=50 donors) and in individual donors with low and high UGT1A3 expression; studies were carried out in the presence and absence of 2% bovine serum albumin (BSA) to also examine the impact of unintended UGT inhibition by free fatty acids released during the incubation1. CDCA phenotyping across 13 recombinant UGTs at 0.2x, 1x and 5x the HLM Km suggested UGT1A1 and UGT2B7 could be contributing to CDCA-24G formation in the liver. Although fm UGT1A1 and UGT2B7 could not be predicted with recombinant data due to lack of a UGT1A3 relative activity factor (RAF), chemical inhibitors were utilized to evaluate their contribution to CDCA-24G formation. Results of these studies demonstrate that while UGT1A3 is the primary metabolic isoform involved in CDCA glucuronidation, UGT2B7 accounts for a significant fraction of CDCA metabolism in the liver, making it a poor -1A3 probe substrate when used alone. While researchers continue to explore and identify other possible UGT1A3 tools, when determining UGT1A3 DDI potential in HLM, it is advised to also include a UGT2B7 selective inhibitor such as β-Phenyllongifolol-2 during incubation.

1. Walsky, R. L., Bauman, J. N., Bourcier, K., Giddens, G., Lapham, K., Negahban, A., Ryder, T. F., Obach, R. S., Hyland, R. and Goosen, T. C. (2012) 'Optimized assays for human UDP-glucuronosyltransferase (UGT) activities: altered alamethicin concentration and utility to screen for UGT inhibitors', Drug Metab Dispos, 40(5), pp. 1051-65.

2. Trottier, J., Verreault, M., Grepper, S., Monte, D., Belanger, J., Kaeding, J., (2006) ‘Human UDP-glucuronosyltransferase (UGT)1A3 enzyme conjugates chenodeoxycholic acid in the liver’, Hepatology, 44(5), pp. 1158–1170.