UGT1A1 and UGT1A3 activity and inhibition in human liver and intestinal microsomes and a recombinant UGT system under similar assay conditions using selective substrates and inhibitors
Creators: name: Mullapudi, T. V. Radhakrishna
name: Ravi, Punna Rao
name: Thipparapu, Ganapathi
Publisher: name: Taylor & Francis
Year: 2021
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Physiology
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Hematology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: 60506 Virology

description: <i>In vitro</i> enzyme kinetics and inhibition data was compared for UGT1A1 and UGT1A3 isoforms under similar assay conditions using human liver microsomes (HLM), human intestinal microsomes (HIM) and recombinant UGT (rUGT) enzyme systems.UGT1A1 catalysed β-estradiol 3-β-D-glucuronide formation showed allosteric sigmoidal kinetics in all enzyme systems; while UGT1A3 catalysed CDCA 24-acyl-β-D-glucuronide formation exhibited Michaelis–Menten kinetics in HLM, substrate inhibition kinetics in HIM and rUGT systems. Corresponding <i>K</i><sub>m</sub> or <i>S</i><sub>50</sub> concentrations of β-estradiol and CDCA were employed in the respective UGT inhibition studies.Atazanavir inhibited the production of β-estradiol 3-β-D-glucuronide with IC50 values of 0.54 µM and 0.16 µM in HLM and rUGT1A1, respectively. But its inhibition potential was not observed in HIM, indicating potential cross-talk with other high-affinity intestinal UGT isozymes. On the other hand, zafirlukast, a pan UGT inhibitor, exhibited moderate inhibition in HIM with an IC50 value of 16.70 µM. Lithocholic acid, inhibited the production of CDCA 24-acyl-β-D-glucuronide with IC50 values of 1.68, 1.84, and 12.42 µM in HLM, rUGT1A3, and HIM, respectively.These results indicated that HLM, HIM, and rUGTs may be used as complementary <i>in vitro</i> systems to evaluate hepatic and intestinal UGT mediated DDIs at the screening stage. <i>In vitro</i> enzyme kinetics and inhibition data was compared for UGT1A1 and UGT1A3 isoforms under similar assay conditions using human liver microsomes (HLM), human intestinal microsomes (HIM) and recombinant UGT (rUGT) enzyme systems. UGT1A1 catalysed β-estradiol 3-β-D-glucuronide formation showed allosteric sigmoidal kinetics in all enzyme systems; while UGT1A3 catalysed CDCA 24-acyl-β-D-glucuronide formation exhibited Michaelis–Menten kinetics in HLM, substrate inhibition kinetics in HIM and rUGT systems. Corresponding <i>K</i><sub>m</sub> or <i>S</i><sub>50</sub> concentrations of β-estradiol and CDCA were employed in the respective UGT inhibition studies. Atazanavir inhibited the production of β-estradiol 3-β-D-glucuronide with IC50 values of 0.54 µM and 0.16 µM in HLM and rUGT1A1, respectively. But its inhibition potential was not observed in HIM, indicating potential cross-talk with other high-affinity intestinal UGT isozymes. On the other hand, zafirlukast, a pan UGT inhibitor, exhibited moderate inhibition in HIM with an IC50 value of 16.70 µM. Lithocholic acid, inhibited the production of CDCA 24-acyl-β-D-glucuronide with IC50 values of 1.68, 1.84, and 12.42 µM in HLM, rUGT1A3, and HIM, respectively. These results indicated that HLM, HIM, and rUGTs may be used as complementary <i>in vitro</i> systems to evaluate hepatic and intestinal UGT mediated DDIs at the screening stage.