Use of in vitro metabolism models for in vivo drug metabolic clearance prediction

• Main Author: Griffin, Michael
• Published: University of Manchester 2007
• Subjects: 615.7
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487998

Recently human liver microsomes have been found to underpredict in vivo human intrinsic clearance (CLint); this has led to an increase in the use of fresh and cryopreserved human hepatocytes as an alternative in vitro system for the prediction of in vivo human CLint. The aim of the work in this thesis was to assess the utility of human hepatocytes for the prediction of in vivo drug metabolic clearance. It was found that fresh and cryopreserved human hepatocytes also underpredict in vivo human CLint (6 and 6 fold respectively). The effect of including the binding to the in vitro system in the clearance prediction was assessed and even though the prediction improved (from 6 fold to 4 fold) the underprediction remained. Investigations were undertaken to explain this underprediction. These involved investigating the metabolism of the probe substrates, tolbutamide and dextromethorphan in different incubation vessels. It was found that there was no significant difference in the CLint of Tolbutamide between 96 well and 24 well plates. The effect of using a preservation solution for the transport of fresh liver tissue was also investigated. Two probe substrates were investigated, namely Midazolam and Clonazepam. It was found that the CLmax of Midazolam in 2 out of the 3 rat livers preserved for one hour in a preservation solution was reduced 2 fold when compared to the CLmax freshly isolated rat hepatocytes. The activity of the CYP3A4 enzyme over the course of a typical metabolism incubation period was also assessed. Studies in cryopreserved human hepatocytes indicated that there was an exponential loss of the CYP3A4 enzyme over the incubation period. The activity of the CYP3A4 enzyme in human liver microsomes remained constant for the first 60 minutes of the incubation. In the case of terfenadine the clearance when measured by the substrate depletion approach was observed to have a biphasic profile in cryopreserved human hepatocytes. This biphasic profile was explained by the loss of the activity of the CYP3A4 enzyme over the incubation period of the assay in cryopreserved hepatocytes. When the CLint was adjusted for the loss of enzyme activity the underprediction was reduced 2 fold. The rate of enzyme activity loss was not apparent in cryopreserved hepatocytes incubated in WME. This may be due to the presence of essential amino acids in WME that are essential for glutathione maintenance in the cell and subsequent protection of the enzyme from reactive oxygen species attack. The underprediction observed in human hepatocytes may be explained by one or a number of the above factors but it most likely that the underprediction is due to a combination of each of the above factors thus efforts to improve the underprediction requires further investigation.