dialysis of the incubate, a suitable (1 : 1) stoichiometry of radioactively labeled
inactivator to active site, and a demonstrable catalytic step for the conversion
of the inactivator to the reactive intermediate (Kent et al., 2001; Silverman,
1988). However, recent reports have suggested that mechanism-based CYP
inactivation does not necessarily conform to 1 : 1 stoichiometry (Koenigs et al.,
1999; Koenigs and Trager, 1998).
Because of the experimental manifestation of time-dependency and require-
ment for CYP catalysis, mechanism-based CYP inactivation is often referred to
as ‘‘time-dependent,’’ ‘‘metabolism-dependent,’’ or ‘‘preincubation-dependent’’
inhibition. A detailed description of the kinetic characteristics of this type of
inhibition has been published (Silverman, 1988) and a simplified kinetic
equation is presented in Table 5.1. In cases where CYP activity can be recovered
by dialysis, the term ‘‘quasi-irreversible’’ inhibition has been proposed (Ma
et al., 2000). In addition, a time-dependency of CYP inhibition can result from
the formation of potent yet reversible metabolites (Ma et al., 2000; Zhao et al.,
2002; Zhout et al., 2005). Formation of a metabolite–intermediate (MI) complex
has been described as another cause for time-dependent CYP inhibition by many
quasi-irreversible inhibitors; in this situation the metabolite or intermediate
coordinates with the heme-ion thus decreasing the rate of catalysis.
5.2.2 In vitroEvaluation of Inhibition
Potential CYP inhibition by new chemical entities (NCEs) can be evaluated
using cDNA-expressed human CYP enzymes (Crespi and Penman, 1997),
human liver microsomes (HLM) (Bjornsson et al., 2003), and cell-based
systems (Yueh et al., 2005a, 2005b;). Within a screening environment, cDNA-
expressed CYP systems with probe substrates that form fluorescent metabolites
have provided the throughput needed to rank order potency and characterize
the structure–activity relationship (SAR), whereas HLM-based assays are
often used for detailed characterization of CYP inhibition and the data are
often included in regulatory submissions. Cell-based assays are used less
frequently due to the lack of availability and reproducibility of cells as well as
factors (cellular uptake or secondary metabolic processes) that may confound
the interpretation of CYP enzyme kinetics. On the contrary, human liver
microsomes and cDNA-expressed CYP enzymes do not represent a true
physiological condition, and therefore, experimental artifacts may arise also. It
is known that discrepancies exist in the generation of inhibition parameters
(e.g., IC 50 or Ki) when using one system over another, and some of the
underlying causes (e.g., nonspecific binding and protein concentration) have
been well described (Margolis and Obach, 2003; Tran et al., 2002; Walsky and
Obach, 2004; Wienkers, 2001; Wienkers and Heath, 2005).
5.2.3 Prediction of CYP Inhibition UsingIn vitroData
Quantitative predictions of in vivo CYP inhibition-mediated drug–drug
interactions have been attempted using mathematical models with various
116 METABOLISM-MEDIATED DRUG–DRUG INTERACTIONS