Abstract
The stilbenic compound combretastatin A-4 (CA-4) has been described as a potent tubulin polymerization inhibitor. In vivo, CA-4 binds to tubulin and inhibits microtubule depolymerization, which results in morphological changes in proliferating endothelial cells. Combretastatin A-4 prodrug phosphate is a leading vascular disrupting agent (VDA), and is currently being evaluated in multiple clinical trials as a treatment for solid tumors. The aim of this study was to identify and characterize the UGT isoforms involved in CA-4 glucuronidation by incubation with human liver microsomes and a panel of nine liver expressed recombinant UGT Supersomes™ (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B15 and 2B17). As we observed, the high rate of formation of CA-4G (Vmax = 12.78 ± 0.29 nmol/min/mg protein) and the low Km (6.98 ± 0.65 μM) denoted that UGT1A9 was primarily responsible for the in vitro glucuronidation of CA-4. UGT1A6 was also a significant contributor to CA-4 glucuronidation (Vmax =3.95 ± 0.13 nmol/min/mg protein and S50 = 44.80 ± 3.54 μM). Furthermore, we demonstrated that the kinetic of CA-4 glucuronidation with liver microsomes but also with a panel of recombinant UGTs is atypical as it fits two different models: the substrate inhibition and also the sigmoidal kinetic model. Finally, experiments conducted to inhibit the glucuronosyltransferase activity in human liver microsomes assay showed that phenylbutazone, trifluoperazine, propofol and 1-naphtol effectively inhibited CA-4 glucuronidation.
- enzyme inhibitors
- glucuronidation
- HPLC
- kinetic modeling
- liver microsomes
- microsomes
- phase II drug metabolism
- polymorphisms
- UDP glucuronyltransferases
Footnotes
- Received December 2, 2009.
- Accepted April 7, 2010.
- The American Society for Pharmacology and Experimental Therapeutics