15.6 Nonradiolabeled Reaction Phenotyping
For the purposes of definition, nonradiolabeled reaction phenotyping is that
conducted ahead of data from radiolabel studies in humans, and is typically
conducted before first dosing of humans (Williams et al., 2003). This differs
from reaction phenotyping later in development, which in the presence of
radiolabel or metabolite standards is more definitive in nature (Bjornsson et al.,
2003). Early reaction phenotyping may employ one or more of the independent
approaches described in Fig. 15.1, whereas late (definitive) reaction phenotyp-
ing is likely to use all three. Early reaction phenotyping has been covered
previously (Williams et al., 2004, 2005), and the essential elements will be
presented in the following sections, with relevant updates.
Perhaps the most important consideration ahead of conduct of experi-
ments is that of downstream value of the data, with particular regard to drug
safety. Compounds that are substrates ofcytochrome P450 enzymes are most
likely to exhibit interindividual variability in pharmacokinetics due to drug–
drug interactions, which would justify a focused effort on cytochrome P450
reaction phenotyping in the early stages of drug discovery (Williams et al.,
2004). Compounds that appear to be substrates of other enzyme systems or
families, such as UGTs (Williams et al., 2004), or esterases are less likely to
be of concerns to safety in patients, and therefore would not necessarily
justify in depth phenotyping of the individual enzymes, especially in the early
stages.
15.6.1 Objective
The objective of early reaction phenotyping is to make a semiquantitative or in
some cases quantitative prediction of the relative contributions of individual
enzymes to the clearance of a compound in humans.
15.6.2 Selection of Appropriate Experimental Systems
15.6.2.1 In silico Advances continue to be made in the prediction of
clearance mechanisms usingin silicoapproaches (Ekins et al., 2001), although
the status of the technology at this time cannot replace the conduct of
experiments in the laboratory.
15.6.2.2 Expressed Enzymes Recombinant P450 enzymes are now available
from multiple vendors in different formats. One major advantage of
recombinant enzymes is that in many preparations the metabolic competence
is significantly higher than in human liver microsomes per milligram protein,
so that the potential for each of the five major cytochrome P450s (CYP1A2,
CYP2C9, CYP2C19, CYP2D6 or CYP3A4) or others to contribute to
the metabolism of the test compound of interest can be rapidly assessed
(Williams et al., 2005). Other advantages include the ability to compare rates of
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