Aims To assess the utility of human hepatic microsomes for predicting in vivo intrinsic clearance (CL int ) via the use of four cytochrome P450 2C9 substrates: phenytoin, tolbutamide (S)-ibuprofen (two pathways) and diclofenac, and to examine the role of exogenous albumin within the microsomal incubation. Methods V max , K m and CL int (defined as V max /K m ratio) were estimated under 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 the microsomal protein index. Non-metabolic related binding in microsomes was measured for phenytoin and tolbutamide in the presence and absence of albumin. Results Microsomal CL int values differed by over two orders of magnitude, with the means ranging from 0.18 ( phenytoin) to 40.70 (diclofenac) ml minmicrosomal protein. When these data were scaled and compared with published in vivo studies a similar rank order was obtained, however, the actual CL int tended to be underpredicted. While the in vivo unbound K m for phenytoin, 1-5 mm is substantially lower than the value determined in microsomes based on total concentrations (56 mm), correction for the in vitro binding reduces this value to 20 mm and 6 mm in the absence and presence of albumin, respectively. Similar trends were seen with tolbutamide K m . Conclusions An appreciation of the utility of in vitro prediction can be best achieved when the range of CL int values predicted from the individual hepatic microsomal samples are compared with the range of individual in vivo CL int values reported in the literature. The degree of underprediction is less evident using the range than the mean data and no consistent advantage in adding albumin to the incubation media is apparent. Keywords: CYP2C9, human microsomes, intrinsic clearance, in vitro predictionsin vitro and in vivo clearances of 25 drugs and were able Introduction to demonstrate a significant correlation despite the numerous sources of these data and the low sample A general strategy to quantitatively predict in vivo drug clearance from CL int values estimated from in vitro drug numbers upon which the parameters were based (see later). There remains a need for more indepth examinmetabolising systems has recently been explored and evaluated in the rat using a data base of 35 drugs which ations of the utility of human in vitro kinetic data for the prediction of the in vivo situation. show a range of turnovers covering four orders of magnitude [1][2][3]. The ultimate application of such aAs there is a shortage of good quality human liver material from which hepatocytes can be successfully prediction strategy is with human tissue, and although there have been many studies with human in vitro tissue, isolated, the vast majority of researchers employ the hepatic microsomal fraction as their enzyme source due often sufficient methodological detail is lacking from the original report to allow a systematic analysis. Iwatsubo to its ease of preparation from tissue of varying quality and i...

A value for human liver microsomal scaling of 40 mg microsomal protein per gram liver has been established. The reason for underprediction previously reported for 52 different drug substrates was not the use of an incorrect value for the scaling factor.

ABSTRACT:Predictions of intrinsic clearance (CL int ) from human liver microsomes often underestimate in vivo observations. In this study, a series of five benzodiazepines was used as prototypic CYP3A4 substrates to investigate the prediction of clearance from the less studied alternative in vitro system, cryopreserved human hepatocytes. Formation of the two major metabolites from midazolam, triazolam, diazepam, flunitrazepam, and alprazolam was measured in cryopreserved human hepatocytes from five donors; the kinetics were characterized and CL int values were determined and scaled to predict CL int in vivo. At least one of the two major pathways of metabolic clearance of each benzodiazepine was characterized by autoactivation in hepatocytes; the extent to which this occurred varied depending on substrate and liver (up to 8-fold). Heteroactivation by testosterone of these pathways was also observed (up to 6-fold). The maximum autoactivated clearance was used to predict in vivo CL int (1.6-138 ml/min/kg) which closely agreed with values previously obtained using human liver microsomes. Comparison with in vivo CL int indicates that cryopreserved human hepatocytes systematically underpredict CL int . When three previous studies (documenting CL int for substrates of various enzymes in cryopreserved human hepatocytes using drug depletion-time profiles) were considered as well, the combined data show a consistent underprediction of 5.6-fold. Collectively it is demonstrated that the predicted hepatic intrinsic clearances from cryopreserved hepatocytes show an excellent rank order with in vivo findings but are systematically underpredicting the in vivo value.

Our study on a group of Jordanians confirmed the well-established observation that broccoli induces CYP1A2 activity. This study also demonstrates the effect of gender and broccoli consumption on CYP2A6 activity in Jordanians.