ABSTRACT:Time-dependent inhibition (TDI) of cytochrome P450 (P450) enzymes caused by new molecular entities (NMEs) is of concern because such compounds can be responsible for clinically relevant drug-drug interactions (DDI). Although the biochemistry underlying mechanism-based inactivation (MBI) of P450 enzymes has been generally understood for several years, significant advances have been made only in the past few years regarding how in vitro time-dependent inhibition data can be used to understand and predict clinical DDI. In this article, a team of scientists from 16 pharmaceutical research organizations that are member companies of the Pharmaceutical Research and Manufacturers of America offer a discussion of the phenomenon of TDI with emphasis on the laboratory methods used in its measurement. Results of an anonymous survey regarding pharmaceutical industry practices and strategies around TDI are reported. Specific topics that still possess a high degree of uncertainty are raised, such as parameter estimates needed to make predictions of DDI magnitude from in vitro inactivation parameters. A description of follow-up mechanistic experiments that can be done to characterize TDI are described. A consensus recommendation regarding common practices to address TDI is included, the salient points of which include the use of a tiered approach wherein abbreviated assays are first used to determine whether NMEs demonstrate TDI or not, followed by more thorough inactivation studies for those that do to define the parameters needed for prediction of DDI.Pharmacokinetic drug-drug interactions (DDIs) can occur when one drug alters the metabolism of a coadministered drug. The outcome is an increase or decrease in the systemic clearance and/or bioavailability, and a corresponding change in the exposure to a coadministered drug. The clinical consequences of DDIs range from lack of therapeutic efficacy of a life saving drug to severe adverse drug reactions, including fatalities. Significant drug-drug interactions can lead to termination of development of otherwise promising new therapies, withdrawal of a drug from the market, or severe restrictions/limitations on its use (Wienkers and Heath, 2005). Because of the impact on patient health and safety, DDI was the subject of a position paper in 2003 by scientists from member companies of the Pharmaceutical Research and Manufacturers of America (PhRMA) that focused on Article, publication date, and citation information can be found at

Nine static models (seven basic and two mechanistic) and their respective cutoff values used for predicting cytochrome P450 3A (CYP3A) inhibition, as recommended by the US Food and Drug Administration and the European Medicines Agency, were evaluated using data from 119 clinical studies with orally administered midazolam as a substrate. Positive predictive error (PPE) and negative predictive error (NPE) rates were used to assess model performance, based on a cutoff of 1.25-fold change in midazolam area under the curve (AUC) by inhibitor. For reversible inhibition, basic models using total or unbound systemic inhibitor concentration [I] had high NPE rates (46-47%), whereas those using intestinal luminal ([I]gut) values had no NPE but a higher PPE. All basic models for time-dependent inhibition had no NPE and reasonable PPE rates (15-18%). Mechanistic static models that incorporate all interaction mechanisms and organ specific [I] values (enterocyte and hepatic inlet) provided a higher predictive precision, a slightly increased NPE, and a reasonable PPE. Various cutoffs for predicting the likelihood of CYP3A inhibition were evaluated for mechanistic models, and a cutoff of 1.25-fold change in midazolam AUC appears appropriate.

The existence of a cytochrome P450-dependent 2,3-epoxide of the potent pneumotoxin 3-methylindole was indirectly confirmed using stable isotope techniques and mass spectrometry. Determination of hydride shift and incorporation of labeled oxygen in 3-methyloxindole and 3-hydroxy-3-methyloxindole, metabolites that may be in part dependent on the presence of the epoxide, were utilized as indicators of the epoxide's existence. One mechanism for the formation of 3-methyloxindole involves cytochrome P450-mediated epoxidation followed by ring opening requiring a hydride shift from C-2 to C-3. Through incubations of goat lung microsomes with [2-2H]-3-methylindole, the retention of 2H in 3-methyloxindole was found to be 81%, indicating a majority of the oxindole was produced by the mechanism described above. 3-Hydroxy-3-methylindolenine is an imine reactive intermediate that could be produced by ring opening of the 2,3-epoxide. The imine may be oxidized to 3-hydroxy-3-methyloxindole by the cytosolic enzyme aldehyde oxidase. Activities of this putative detoxification enzyme were determined in both hepatic and pulmonary tissues from goats, rats, mice, and rabbits, but the activities could not be correlated to the relative susceptibilities of the four species to 3-methylindole toxicity. The 18O incorporation into either 3-methyloxindole or 3-hydroxy-3-methyloxindole from both 18O2 and H218O was determined. The 18O incorporation into 3-methyloxindole from 18O2 was 91%, strongly implicating a mechanism requiring cytochrome P450-mediated oxygenation. Incorporation of 18O into 3-hydroxy-3-methyloxindole indicated that the alcohol oxygen originated from molecular oxygen, also implicating an epoxide precursor. These studies demonstrate the existence of two new reactive intermediates of 3-methylindole and describe the mechanisms of their formation and fate.

Zafirlukast is a leukotriene antagonist indicated for the treatment of mild to moderate asthma, but the drug has been associated with occasional idiosyncratic hepatotoxicity. Structurally, zafirlukast is similar to 3-methylindole because it contains an N-methylindole moiety that has a 3-alkyl substituent on the indole ring. The results presented here describe the metabolic activation of zafirlukast via a similar mechanism to that described for 3-methylindole. NADP(H)-dependent biotransformation of zafirlukast by hepatic microsomes from rats and humans afforded a reactive metabolite, which was detected as its GSH adduct. Mass spectrometry and NMR data indicated that the GSH adduct was formed by the addition of GSH to the methylene carbon between the indole- and methoxy-substituted phenyl rings of zafirlukast. The formation of this reactive metabolite in human liver microsomes was shown to be exclusively catalyzed by CYP3A enzymes. Evidence for in vivo metabolic activation of zafirlukast was obtained when the same GSH adduct was detected in bile of rats given an iv or oral dose of the drug. On the basis of results with model peroxidases and of the structures of product alcohols from incubations containing H2(18)O, it appeared that zafirlukast underwent dehydrogenation by two sequential one-electron oxidations. In addition, zafirlukast proved to be a mechanism-based inhibitor of CYP3A4 activity in human liver microsomes and in microsomes containing cDNA-expressed CYP3A4. The enzyme inhibitory property of zafirlukast was selective for this enzyme among all of the P450 enzymes that were tested in human liver microsomes. The inactivation was characterized by a K(I) of 13.4 microM and k(inact) of 0.026 min(-1). In summary, zafirlukast dehydrogenation to an electrophilic alpha,beta-unsaturated iminium intermediate may be associated with idiosyncratic hepatotoxicity and/or cause drug-drug interactions through inactivation of CYP3A4.