DRF-4367 is a novel
COX-2 inhibitor, which showed good efficacy in several animal models of
inflammation. In a comparative in vitro metabolism in various liver microsomes,
DRF-4367 forms a hydroxy metabolite (DRF-6574) mediated by
CYP2D6 and 2C19
isoenzymes.
DRF-6574 readily undergoes Phase-II metabolism and forms
glucuronide and
sulfate conjugates both in vitro and in vivo. The objective of the present study was two folds: to study the glucuronidation of
DRF-6574 in human liver and intestinal microsomes and to identify the recombinant human liver and intestinal
UDP-glucuronosyltransferase (UGT)
enzymes responsible for glucuronidation of
DRF-6574. Of twelve recombinant UGTs tested, two hepatic UGTs viz., UGT1A1 and 1A3 and an extra hepatic UGT i.e.,
UGT1A8 showed the catalytic activity. The
enzyme kinetics in pooled human liver, intestinal and recombinant UGT microsomes showed a typical Michaelis-Menten plot. The apparent Km and Vmax value for
DRF-6574 was found to be 116 +/- 24 microM and 2.07 +/- 0.12 microg/min/mg
protein and 142 +/- 17 microM and 3.83 +/- 0.15 microg/min/mg
protein in pooled human liver and intestinal microsomes, respectively. The intrinsic clearance (Vmax/Km) value for
DRF-6574 was estimated to be 0.043 and 0.065 ml/min/mg
protein, respectively in pooled human liver and intestinal microsomes. Moreover we have determined the Km and Vmax and intrinsic clearance values for specific UGTs viz., UGT 1A1, 1A3 and 1A8. The apparent Km and Vmax values are 23 +/- 7.2 microM, 3.44 +/- 0.17 microg/min/mg
protein for UGT1A1, 60 +/- 7.9 microM, 3.67 +/- 0.11 microg/min/mg
protein for UGT1A3, 96 +/- 8.0 microM, 2.95 +/- 0.06 microg/min/mg
protein for
UGT1A8. The intrinsic clearance values (Vmax/Km) estimated were 0.367, 0.148, 0.074 ml/min/mg
protein for UGT1A1, 1A3 and 1A8, respectively. The intrinsic clearance value in
UGT1A8 was very close to that in human intestinal and liver microsomes. The formation of
DRF-6574 glucuronide by human liver, intestinal and UGT1A1, 1A3 and 1A8 microsomes was effectively inhibited by
phenylbutazone.