Comparison of Relative Activity versus Relative Expression Factors (RAF versus REF) in Predicting Glucuronidation...

Area of Science:

• Drug metabolism and pharmacokinetics
• Enzyme kinetics
• Biotechnology

Background:

• Predicting the quantitative fraction of glucuronidation (f_gluc) by individual UDP-glucuronosyltransferase (UGT) enzymes is crucial for drug development.
• Challenges exist due to the lack of selective inhibitors and inconsistent activity of recombinant UGT systems (rUGTs).

Purpose of the Study:

• To compare relative expression factors (REF) versus relative activity factors (RAF) for predicting f_gluc.
• To assess the utility of REF and RAF using rUGT data against human liver and intestinal microsomes (HLM and HIM).

Main Methods:

• REF scalars were derived from proteomics data for eleven UGT enzymes.
• RAF was calculated by measuring activities in rUGTs and microsomes with selective UGT probe substrates.
• Protein-normalized activity factor (pnAF) was generated by correcting UGT activity to protein abundance.

Main Results:

• REF values ranged from 0.02 to 1.75; RAF values ranged from 0.1 to 274.
• pnAF values were generally 5- to 80-fold, with exceptions for UGT2B4 (~180-fold) and UGT2B15 (>1000-fold).
• Differential specific activities of rUGTs confounded f_gluc predictions.

Conclusions:

• Recombinant UGT enzyme activity is significantly lower than in microsomes at equivalent protein amounts.
• Poor and variable specific activity of rUGTs (per pmol protein) necessitates careful consideration in f_gluc scaling.