Effect of Albumin on Human Cytochromes P450 Kinetics: Extrapolation of in Vivo Clearance from in Vitro Data

Abstract

It has been reported that addition of bovine serum albumin (BSA) to in vitro
incubations improves estimation of kinetic parameters, intrinsic clearance (CLint = Vmax/Km),
by decreasing the Km for drugs metabolised by cytochrome P450 2C9 (CYP2C9). However,
the effect of BSA on other CYP isozymes was unclear. The aims of this study were to
characterize effect of BSA on kinetics of specific pathways for CYP2C8, 2C19, and 3A4
whether the addition of BSA could improve the prediction of in vivo clearance by using
human hepatic microsomal 6α-hydroxypaclitaxel, 5-hydroxyomeprazole, and omeprazole
sulfone formations as markers for CYP2C8, 2C19, and 3A4 pathways, respectively. The
metabolite formations were determined by HPLC. In the presence of 2% BSA, rate of
CYP2C8-mediated 6α-hydroxypaclitaxel formation was well described by single enzyme
Michaelis-Menten kinetic. Mean CLint was significantly increased about 4 fold. Thus the
extrapolation of in vitro CLint to in vivo clearance for paclitaxel 6α-hydroxylation was more
accurate in the presence of BSA in the incubation. 5-Hydroxyomeprazole formation in the
presence of 2% BSA followed two enzyme Michaelis-Menten kinetics. In the presence of 2%
BSA, the mean Km1 and Vmax1 for omeprazole 5-hydroxylation were decreased 4 and 2 fold,
respectively which resulted in 2-fold increases in the mean CLint1. Moreover, the mean Km
and Vmax for CYP3A4-mediated omeprazole sulfoxidation were increased (15-19 fold) in the
presence of 2% BSA therefore the mean CLint was not significantly changed. BSA converted
omeprazole sulfoxidation from two enzyme to single enzyme Michaelis-Menten kinetic. In
conclusion, the addition of 2% BSA is likely to improve in vitro clearance prediction for
CYP2C8-mediated paclitaxel 6α-hydroxylation. Moreover, BSA showed minor effect on the
CYP2C19-mediated omeprazole 5-hydroxylation whereas had no effect on CYP3A4-
mediated omeprazole sulfoxidation. The effect of albumin on individual CYP isoforms was
variable; the use of BSA to improve prediction of in vivo intrinsic clearance seems to be
possible with only CYP2C8.