Abstract

An in vitro glucuronidation assay was used to optimize a series of N-hydroxyurea-containing 5-lipoxygenase inhibitors for metabolic stability. The glucuronidation of these compounds in cynomolgus monkey microsomes followed Michaelis-Menten kinetics allowing calculation of V_max andK_M . The V_max values ranged from 0.02 to 7.9 nmol/min/mg microsomal protein, a 400-fold difference, whereas K_M ranged from 204 to 2500 μM, only a 12-fold difference. In vitro intrinsic clearance values (CL_int) were calculated for 18 compounds tested in the kinetic assay and compared with the in vivo plasma clearance (CL_p ) calculated from intravenous studies done in cynomolgus monkeys. These initial results suggested a relationship between the in vitro CL_int and in vivo duration as defined byCL_p . A more rapid in vitro assay was developed in a 96-well format using a single concentration of substrate (100 μM) from which a glucuronidation rate was calculated. The results from this assay for 40 compounds correlated with in vivo plasma clearance (r = 0.57). This more efficient assay was used to test more than 100 compounds and develop structure–metabolism relationships based on metabolic stability and improved duration. The culmination of this effort contributed to the discovery of ABT-761, a 5-lipoxygenase inhibitor with in vivo duration in monkey improved 40-fold over the first generation inhibitor. Further studies performed in human liver microsomes demonstrated a similar trend that was corroborated by the 8-fold increase in duration after oral dosing in humans observed with ABT-761.