Evaluation of Time-Dependent Cytochrome P450 Inhibition Using Cultured Human Hepatocytes

Abstract

ABSTRACT:Primary human hepatocytes in culture are commonly used to evaluate cytochrome P450 (P450) induction via an enzyme activity endpoint. However, other processes can confound data interpretation. To this end, the impact of time-dependent P450 inhibition in this system was evaluated. Using a substrate-cassette approach, P450 activities were determined after incubation with the prototypic inhibitors tienilic acid (CYP2C9), 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 recombinant P450s and human liver microsomes (HLMs). 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. ) effectively abolished CYP2C9 activity over 24 h at low (micromolar) 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 P450 activity in this system. Therefore, in addition to enzyme activity, mRNA and/or protein levels should be measured to fully evaluate the P450 induction potential of a drug candidate.Inhibition of cytochrome P450 (P450)-dependent metabolism is a prevalent source of drug-drug interactions (DDIs) and may result in serious clinical consequences (Jankel and Fitterman, 1993; Bertz and Granneman, 1997) via either reversible or irreversible means. An assessment of the potential of a new chemical entity to cause DDIs via inhibition of P450 metabolism is important early in the drug discovery process. Mechanism-based P450 inhibitors can be characterized as displaying NADPH-, concentration-, and time-dependent quasi-irreversible or irreversible inactivation because of chemical modification of the heme and/or protein, and the inactivation rate is diminished in the presence of a competing substrate (Silverman, 1998). Such inhibitors may be of specific concern; first because the in vivo inhibitory effect lasts longer than that for reversible inhibitors and inactivated P450 has to be replaced by newly synthesized protein; and second because of an increased toxicological risk via the generation of reactive species (Zhou et al., 2005).Many drugs are mechanism-based inhibitors of P450, including the macrolide antibiotics erythromycin and troleandomycin (Larrey et al., 1983), tienilic acid (Lopez-Garcia et al., 1994), and fluoxetine (Mayhew et al., 2000). Thus, in vitro screens to determine both the degree and nature of P450 inhibition (typically determining the time dependence of inhibition) are now used across the pharmaceutical industry. More often than not, these assays focus on the five major drugme...

Sequential Metabolism of Secondary Alkyl Amines to Metabolic-Intermediate Complexes: Opposing Roles for the Secondary Hydroxylamine and Primary Amine Metabolites of Desipramine, (S)-Fluoxetine, and N-Desmethyldiltiazem

“…Fluoxetine causes TDI of CYP2C19 activity in Supersomes, HLMs, hepatocytes, and profound inhibition in vivo (McGinnity et al, 2006). Thus, we conclude that the inactivation of CYP2C19 both in vitro and most likely in vivo is due to formation of the secondary hydroxylamine metabolite of fluoxetine, which subsequently forms an MI complex to inactivate the enzyme.…”

“…Further characterization of alkyl amine metabolism by P450 enzymes in single enzyme and multiple enzyme preparations is necessary before establishment of guidelines for prediction. It seems prudent to include studies in hepatocytes, in which the metabolites are exposed to the full complement of enzymes, when seeking to eliminate or confirm the possibility of DDIs with alkyl amines (Zhao et al, 2005;McGinnity et al, 2006). Levels of primary amines and hydroxylamines in vitro and in vivo may be significantly different, and the prediction of primary and secondary metabolite concentrations in vivo from in vitro data is problematic at best and warrants further investigation.…”

In Vitro Evaluation of Metabolic Drug–Drug Interactions: Concepts and Practice

“…Time-dependent inhibition of P450 can also be studied using intact human hepatocytes (McGinnity et al, 2006). One precaution with the use of intact hepatocytes is to concurrently measure also cytotoxicity.…”

Integrated in Vitro Analysis for the in Vivo Prediction of Cytochrome P450-Mediated Drug-Drug Interactions

“…The interaction of ritonavir with desipramine (observed ␦AUC 2.5) was somewhat underpredicted using [I] in,u /K i (␦AUC 1.4) and Simcyp (␦AUC 1.2, range 1.1-1.5), whereas the interaction of fluoxetine with all the substrates was significantly underpredicted using [I] in,u /K i (Table 4). Using Simcyp to simulate the in vivo contribution of the major human metabolite of fluoxetine, norfluoxetine, by inputting the rP450 K i values (Table 1), the mechanismbased inhibition of CYP3A4 and CYP2C19 by fluoxetine (Mayhew et al, 2000;McGinnity et al, 2006), and using the maximum active uptake factor of 1000 of both drug and metabolite into the liver resulted in a good estimation of the degree of interaction with imipramine [observed ␦AUC 3.3 versus simulated ␦AUC 3.8 (2.0 -7.7)], but the interaction with desipramine was still somewhat underestimated (Table 4). The measured IC 50,u values of fluoxetine, norfluoxetine, fluvoxamine, ritonavir, labetalol, or sertraline for CYP2D6 did not significantly alter between rP450 and cryopreserved human hepatocytes.…”