Received for publication April 7, 2004.
Revised June 8, 2004.
Accepted for publication June 8, 2004.

USE OF THE SUBSTRATE DEPLETION APPROACH FOR DETERMINING IN VITRO METABOLIC CLEARANCE: TIME DEPENDENCIES IN HEPATOCYTE AND MICROSOMAL INCUBATIONS

Abstract

The substrate depletion method is a popular approach used for the measurement of in vitro intrinsic clearance (CL_int). However the incubation conditions used in these studies can vary; the consequences of which have not been systematically explored. Initial substrate depletion incubations using rat microsomes and hepatocytes were performed for eight benzodiazepines: alprazolam, clobazam, clonazepam, chlordiazepoxide, diazepam, flunitrazepam, midazolam and triazolam. Subsequent predictions of in vivo CL_int (ranged from 3-200ml/min) and hepatic clearance (CL_H) (ranged from 0.3-15ml/min) demonstrated that the general predictive ability of this approach was similar to the traditional metabolite formation method. A more detailed study of the substrate depletion profiles and CL_int estimates indicated that the concentration of enzyme used is of particular importance. The metabolism of triazolam, clonazepam and diazepam was monoexponential at all cell densities using hepatocytes; however with microsomes, biphasic depletion was apparent particularly at higher microsomal protein concentrations (2-5mg/ml). Enzyme activity studies indicated that enzyme loss was more pronounced in the microsomal system (ranged from 8-65% activity after a 1 hour incubation) compared to the hepatocyte system (approximately 100% activity after a 1 hour incubation). For clonazepam (a low clearance substrate) these biphasic profiles could be explained by loss of enzyme activity. To ensure accurate predictions of in vivo CL_int and CL_H when using the substrate depletion approach, based on the results obtained for this class of drugs, it is recommended that low enzyme concentrations and short incubation times are used whenever possible.

Key words: enzyme kinetics, in vitro-in vivo prediction, isolated hepatocytes, liver microsomes, metabolite kinetics

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