Drug Metabolism in Drug Design and Development Basic Concepts and Practice

spectrometer to waste for the first and last minute of the gradient to remove
nonvolatile salts. Mass to charge value for each metabolite formed is shown in
Table 16.1. For quantitation of the metabolites, the mass spectrometer was
operated in the MRM modes to monitor area ratios of metabolite(s) to internal
standard.

16.3.4 Data Analysis

The observed rates of individual CYP inactivations (kobs) are calculated from the
initial slopes of the linear regression lines of semilogarithmic plots (natural
logarithm of remaining activity versus preincubation time, Fig. 16.6a).kinactand
KIcan be obtained by nonlinear regression using Equation 16.5 (Fig. 16.6b). In
most cases, the parameters are also generated by the double reciprocal
Lineweaver–Burk plot (1/kobsversus 1/[I]) as depicted in Fig. 16.6c (Eq. 16.6),
which showskinactestimate at reciprocal of they-intercept andKIat negative
reciprocal of thex-intercept.

kobs¼

kinact½IŠ

KIþ½IŠ

ð 16 : 5 Þ

1

kobs

¼

KI

kinact

1

½IŠ

þ

1

kinact

ð 16 : 6 Þ

16.4 Fluorescent Assay

The assays with fluorescence detection in a HTS manner have been employed
in pharmaceutical companies to determine the CYP inhibition potential of the
drug candidates. These assays are usually applied for major human CYPs, such
as 1A2, 2C9, 2C19, 2D6, and 3A4, in which a fluoroprobe, through a CYP-
mediated biotransformation, becomes fluorescent (Crespi et al., 2000; Donato
et al., 1998; Kennedy and Jones, 1994). These fluoroprobes may not be
selective for an individual CYP, and thus the recombinant cDNA-expressed
CYPs in microsomal preparations have to be used (Crespi et al., 2000; Kariv
et al., 2001; Trubetskoy et al., 2005). The fluorescent HTS has been well
developed into 96-well, 384-well, or even higher microplate format (Crespi
et al., 2000; Kariv et al., 2001; Kennedy and Jones, 1994; Trubetskoy et al.,
2005). The technology has been further developed into cell-based investigation,
where CYPs are transfected into and expressed in cell lines (Crespi and
Stresser, 2000; Donato et al., 2004). Microarray technology, combining
solidified enzymes and fluorescence, has been recently reported for HTS
(Sakai-Kato et al., 2005). The major fluorescence HTS assays for CYP
inhibition are summarized on Table 16.5.
The better fluoroprobes would produce strong fluorescent metabolite with
higher excitation (>400 nm) and emission (>500 nm) wavelengths to reduce

532 ANALYSIS OFIN VITROCYTOCHROME P450 INHIBITION