Microsomal prediction of in vivo clearance of CYP2C9 substrates in humans

Aims To assess the utility of human hepatic microsomes for predicting in vi vo intrinsic clearance (CLint) via the use of four cytochrome P450 2C9 subs trates: phenytoin, tolbutamide (S)-ibuprofen (two pathways) and diclofenac, and to examine the role of exogenous albumin within the microsomal incubat ion. Methods V-max, K-m and CLint (defined as V-max/K-m ratio) were estimated un der initial rate conditions for five pathways of metabolism in a bank of 15 human hepatic microsomal samples and were scaled to in vivo units using th e microsomal protein index. Non-metabolic related binding in microsomes was measured for phenytoin and tolbutamide in the presence and absence of albu min. Results Microsomal CLint values differed by over two orders of magnitude, w ith the means ranging from 0.18 (phenytoin) to 40.70 (diclofenac) mu l min( -1) mg(-1) microsomal protein. When there data were scaled and compared wit h published in vivo studies a similar rank order was obtained, however, the actual CLint tended to be underpredicted. While the in vivo unbound K-m fo r phenytoin, 1-5 mu M is substantially lower than the value determined in m icrosomes based on total concentrations (56 mu M), correction for the in vi tro binding reduces this value to 20 mu M and 6 mu M in the absence and pre sence of albumin, respectively. Similar trends were seen with tolbutamide K -m. Conclusions An appreciation of the utility of in vitro prediction can be be st achieved when the range of CLint values predicted from the individual he patic microsomal samples are compared with the range of individual in vivo CLint values reported in the literature. The degree of underprediction is l ess evident using the range than the mean data and no consistent advantage in adding albumin to the incubation media is apparent.