Radioisotope-based inhibition assays have been also developed for the CYP
inhibition using probe substrates, in which the O- or N-alkyl group contained
either tritium or^14 C-labeled (Draper et al., 1998a, 1998b; Di Marco et al.,
2005; Moody et al., 1999; Riley and Howbrook, 1997). The assays do offer
some advantages over the fluorometric assays including substrate selectivity,
better solubility of substrates, and higher rates of substrate turnover. However,
the major disadvantage of the assays is that they require a separation
procedure, such as solid-phase extraction, for separating metabolites from the
parent compound before analysis, which limits these approaches inadequate
for HTS. An LC/MS method with specific drug probes appears to overcome
many of these limitations by above two methods (Cohen et al., 2003; Bu et al.,
2001a, 2001b; Nomeir et al., 2001; Yin et al., 2000; Zhang et al., 2002). It can
provide sufficient throughput (automated 96-well plate and fast direct online
injection to LC/MS/MS), flexibility (variety of probe substrate and enzyme
source, i.e., recombinant CYPs and HLM), and instrument selectivity,
sensitivity, and speed (rapid gradient LC and simultaneous detection of
multiple metabolites). For example, the LC/MS uses both ballistic gradient
liquid chromatography and mass spectrometry for quantification of the drug
and the metabolite(s). Mass-spectrometric analysis using single ion monitoring
(SIM) provides high sensitivity and eliminates the need for long gradient for
the resolution of the peaks during liquid chromatography. In addition, a new
and robust method for the simultaneous evaluation of the multiple CYP
enzyme activities in HLM has been also accomplished, using rapid gradient
LC/MS with SRM of specific metabolites (Bu et al., 2001a, 2001b; Dierks et al.,
2001). In addition to the above CYP inhibition assays, automation and
validation of the assays following good laboratory practice (GLP) level have
been largely dealt and developed, thereby leading to data with high quality and
precise (Jenkins et al., 2004; Kremers 2002; Walsky and Obach, 2004; Yao
et al., 2007). The focus of this chapter is to provide theoretical background for
enzyme inhibition kinetics (reversible and irreversible), general methods for
determining the inhibition kinetic parameters, inhibition assays specific to
individual CYPs with LC/MS/MS approaches, and available tools and
guidelines for prediction of DDIs in human from thein vitroinhibition data.
16.2 Reversible Inhibition
When an inhibitor is added to the enzyme reaction, the reaction mixture
may comprise more than one enzyme complex, namely ES, EI, and/or ESI
(Scheme 16.1) (Segel, 1987; Shou et al., 2001). Since the ES concentration
([ES]) decreases with an increase in [I], the rate of product formation (kp[ES])
can decline (0b<1). Scheme 16.1 depicts a general kinetic model used to
describe the interaction between substrate (S), inhibitor (I) and enzyme (E).
Based on nature of inhibition, inhibition kinetics can be categorized to
competitive, noncompetitive, uncompetitive and mixed type inhibitions.
REVERSIBLE INHIBITION 515