Prediction of Glucuronidation (UGT) clearance using alamethicin activated human liver microsomes

An increasing number of compounds are metabolised via a direct phase II pathway (e.g., glucuronidation) in parallel to elimination by phase I enzymes. Therefore, there is a need to evaluate the use of microsomes for the prediction of clearance for such compounds. In this study the clearance for nine compounds with differential contribution of CYP and UGT pathways was determined. In order to investigate the importance of metabolism via glucuronidation the intrinsic clearance (CLint) was determined in the presence of either CYP (NADP) or UGT (UDPGA) cofactors, both individually and combined. For incubations with UDPGA human liver microsomes were activated with alamethicin at a concentration of 50 μg/mg protein. In the presence of UDPGA the CLint,UGT ranged from 8.0 to 479 μL/min/mg protein for ketoprofen and raloxifene, respectively. In the presence of NADP the CLint,CYP ranged from 3.9 to 3056 μL/min/mg protein for naloxone and propofol, respectively. The CLint with combined cofactors present was approximately additive when compared to the sum of the individual CLint,CYP and CLint,UGT. In the presence of both cofactors the CLint ranged from 13.0 to 3355 μL/min/mg protein for naloxone and propofol, respectively. For each of the compounds investigated the fraction metabolised by UGT (fmUGT) and CYP (fmCYP) enzymes was calculated from the individual cofactor data. The in vitro clearance data indicated a varying contribution of glucuronidation to the clearance of the compounds studied with the fmUGT ranging from 0.09 to 0.83 for propofol and gemfibrozil, respectively. The CLint values obtained from incubations were corrected for nonspecific microsomal binding and scaled using a human microsomal scaling factor of 40 mg/g protein and an average liver weight of 21 g liver/kg. A good correlation between the predicted and observed glucuronidation CLint was obtained using data with only UDPGA present. In addition, predicted CLint obtained from data in the presence of both cofactors correlated well with the observed total CLint. The current study indicates the applicability of both individual and combined cofactor conditions in the assessment of clearance for compounds with a differential contribution of CYP and UGT enzymes to their elimination.