Effect of phenobarbital and p-hydroxyphenobarbital glucuronide on acetaminophen metabolites in isolated rat hepatocytes: use of a kinetic model to examine the rates of formation and egress.

Abstract

Conventional analysis of initial uptake and egress rates in isolated hepatocytes is limited in the ability to distinguish between rates of metabolite formation and egress, and to separate basolateral and canalicular transport processes. The present study examined the applicability of kinetic modeling in describing acetaminophen glucuronide (AG) and acetaminophen sulfate (AS) formation and egress in hepatocytes after acute exposure to phenobarbital or p-hydroxyphenobarbital glucuronide (p-OHPBG) in vitro, or in vivo phenobarbital pretreatment. A significant pretreatment effect on AG and AS disposition was seen based on initial rates of egress. In vivo phenobarbital pretreatment decreased the initial egress rate of AG compared to vehicle pretreatment, and the initial egress rate of AS compared to all other treatments. A pharmacokinetic model incorporating AG and AS formation in hepatocytes as well as egress processes (including diffusional and active transport components) was fit to the data. Parameter estimates derived from model fits to the data showed the expected increase in acetaminophen glucuronidation and decrease in sulfation after phenobarbital pretreatment; in addition, an increase in the AG diffusional rate constant and a decrease in the AS diffusional rate constant was apparent. The excretion Vmax for AG was decreased statistically after acute phenobarbital exposure in vitro, and in vivo phenobarbital pretreatment, with a concomitant statistical increase in the Km for AG excretion. In vitro acute p-OHPBG exposure also decreased significantly the excretion Vmax for AG. These data are consistent with the hypothesis that phenobarbital-impaired biliary excretion of AG is a function of impaired canalicular transport due to the presence of p-OHPBG. They further suggest that the mechanism may not be simple competitive inhibition. This work demonstrates the utility of a kinetic modeling approach to differentiate metabolic and transport processes when analyzing data from isolated hepatocyte studies. Additional information may be gained that would not be apparent by conventional methods of analysis.