Enzyme Kinetics of Uridine Diphosphate Glucuronosyltransferases (UGTs)

Abstract

Glucuronidation, catalyzed by uridine diphosphate glucuronosyltransferases (UGTs), is an important process for the metabolism and clearance of many lipophilic chemicals, including drugs, environmental chemicals, and endogenous compounds. Glucuronidation is a bi-substrate reaction that requires the aglycone and a cofactor, UDPGA. Accumulating evidence suggests that the bi-substrate reaction follows a compulsory-order ternary mechanism. To simplify the kinetic modelling of glucuronidation reactions in vitro, UDPGA is usually added to incubations in large excess. Many factors have been shown to influence UGT activity and kinetics in vitro, and these must be accounted for in experimental design and data interpretation. Assessing drug–drug interactions (DDIs) involving UGT inhibition remains challenging. However, the increasing availability of UGT enzyme-specific substrate and inhibitor “probes” provides the prospect for more reliable reaction phenotyping and assessment of DDI potential. Although extrapolation of the in vitro intrinsic clearance of a glucuronidated drug often under-predicts in vivo clearance, careful selection of in vitro experimental conditions and inclusion of extrahepatic glucuronidation may improve the predictivity of in vitro–in vivo extrapolation (IVIVE).

Appendices

Appendix: A Representative Protocol on In Vitro Glucuronidation: Determination of Kinetic Parameters for Estradiol-3-Glucuronidation in HLM

The following protocol illustrates the incubation conditions and procedures for determining the kinetic parameters of estradiol-3-glucuronidation in HLM. Based on prior time and protein linearity studies, the kinetic characterization is conducted with 0.25 mg/mL of protein and for an incubation time of 20 min.

Reagents

• Pooled human liver microsomes (BD Biosciences; Woburn, MA)

• UltraPure™ 1 M Tris–HCl, pH 7.4, (Invitrogen; Carlsbad, CA)

• Magnesium chloride (MgCl₂) hexahydrate (Sigma; St. Louis, MO)

• Alamethicin (Sigma; St. Louis, MO)

• β-Estradiol and estadiol-3-glucuronide (Sigma; St. Louis, MO)

• 4-Methylumbelliferyl-β-d-glucuronide (4-MUG) hydrate (Sigma; St. Louis, MO) as internal standard

Procedures

• 1. Prepare the following stock solutions

  • Incubation buffer: 1 mM MgCL₂ in Tris–HCl Buffer (0.05 M, pH = 7.4)

  • 5 mg/mL alamethicin in ethanol

  • 50 mM UDPGA in incubation buffer

  • Estradiol stock solution: 8–10 concentrations; the concentrations of the stock solutions are 100-fold higher than the actual concentrations in the incubation.

• 2. Prepare alamethicin-activated HLM

  Dilute pooled HLM (20 mg/mL) with incubation buffer, and then add alamethicin (5 mg/mL). The final protein concentration and alamethicin content are 0.625 mg/mL and 50 μg/mg of protein, respectively. Incubate the resulting mixture on ice for 15–30 min.

• 3. Mix all the incubation components, except UDPGA, according to Table 2, and then incubate the resulting mixture and UDPGA stock solution separately in a 37 °C water bath for 3 min.

  Table 2 Incubation components and the volume of each component added to the incubation

• 4. Add pre-warmed UDPGA stock solution to start the reaction.

• 5. After incubating the complete incubation mixture at 37 °C for 20 min, stop the reaction by adding equal volume of acetonitrile, containing the internal standard, 0.5 μM 4-methylumbelliferone glucuronide.

• 6. Centrifuge the resulting mixture at 13,000 × g for 5 min. The concentrations of estradiol-3-glucuronide in the supernatants are determined by LC-MS/MS analyses, based on standard curves prepared with an authentic estradiol-3-glucuronide reference standard.