Received for publication February 24, 2006.
Revised April 18, 2006.
Accepted for publication April 25, 2006.

Evaluation of time-dependent cytochrome P450 inhibition using cultured human hepatocytes

Abstract

Primary human hepatocytes in culture are commonly used to evaluate CYP induction via an enzyme activity endpoint. However, other processes can confound data interpretation. To this end, the impact of time-dependent CYP inhibition in this system was evaluated. Using a substrate-cassette approach, CYP activities were determined after incubation with the prototypic inhibitors tienilic acid (CYP2C9) and erythromycin, troleandomycin and fluoxetine (CYP3A4). Kinetic analysis of enzyme inactivation in hepatocytes was used to describe the effect of these time-dependent inhibitors and derive the inhibition parameters k_inact and K_I, which generally were in good agreement with the values derived using rCYPs and HLM. Tienilic acid selectively inhibited CYP2C9 dependent diclofenac 4'-hydroxylation activity and erythromycin, troleandomycin and fluoxetine inhibited CYP3A4 dependent midazolam 1'-hydroxylation in a time- and concentration dependent manner. Fluoxetine also inhibited CYP2C19 dependent S-mephenytoin 4'-hydroxylation in a time- and concentration-dependent manner in hepatocytes, HLM and rCYP2C19 (K_I 0.4 µM and k_inact 0.5 min^-1). As expected, the effect of fluoxetine on CYP2D6 in hepatocytes was consistent with potent yet reversible inhibition. A very weak time-dependent CYP2C9 inhibitor (AZ1, K_I 30 µM & k_inact 0.02 min^-1) effectively abolished CYP2C9 activity over 24 h at low (µM) concentrations in primary cultured human hepatocytes. This work demonstrates that caution is warranted in the interpretation of enzyme induction studies with metabolically stable, weak time-dependent inhibitors, which may have dramatic inhibitory effects on CYP activity in this system. Therefore, in addition to enzyme activity, mRNA and/or protein levels should be measured in order to fully evaluate the CYP induction potential of a drug candidate.

Key words: bioactivation, CYP induction, CYP inhibition, cytochrome P450, drug discovery, drug-drug interactions, hepatocytes, mechanism-based inhibition

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