pathways (such as CYP3A4 by ketoconazole) would render this subpopulation
at a much greater risk for adverse events when a drug is cleared largely via two
CYPs only (e.g., CYP2C19 and CYP3A4). Therefore, CYP reaction phenoty-
ping data are extremely useful when predicting pharmacokinetic-based DDIs,
designing clinical studies, and understanding pharmacokinetic variability in
human subjects.
Ideally, definitive CYP reaction phenotyping should be available before
the initiation of clinical development. Unfortunately, more accurate data
can only be obtained once clearance pathways are identified in human subjects,
and human radiolabeled studies are generally not conducted as the first set
of clinical studies for NCEs. In this context, CYP reaction phenotyping, per-
formed using various humanin vitrosystems is expected to be as complete as
possible (Bjornsson, 2003).
Figure 5.2 describesin vitro reaction phenotype data for five CYP3A4
substrates that have gone into development. In each case, the impact of
ketoconazole on their pharmacokinetics has been evaluated clinically. As
expected, CYP3A4 played a major role in the overall clearance of compounds
‘‘C,’’ ‘‘D,’’ and ‘‘E’’ and ketoconazole elicited a marked impact on their
pharmacokinetics (>4-fold increase in AUC). In comparison, the enzyme
played a minor role in the clearance of compounds ‘‘A’’ and ‘‘B’’ and the AUC
increase due to ketoconazole was less marked (<2-fold).
5.4.1 Experimental Considerations
During drug discovery and early development, initial CYP-reaction phenoty-
ping studies are generally conducted using cDNA-expressed human CYP
systems by monitoring substrate depletion, due to the lack of synthetic
metabolites or radiolabeled material. Results from this type of study often yield
useful and qualitative information related to the CYP(s) responsible for
metabolic clearance. Once synthetic standards of metabolites become available,
cDNA-expressed human CYP systems can then be optimized and definitive
kinetic parameters such as Km andVmax(orkcat) can be determined by
monitoring metabolite formation. The choice of substrate concentration range
is an important consideration in early CYP reaction phenotyping studies
because one has to ensure that the experimental conditions enable the
identification of lowKm( 20 mM) CYP forms. In addition to cDNA-expressed
human CYP enzymes, metabolism of NCEs can be investigated with human
liver microsomes in the presence of specific chemical inhibitors or inhibitory
antibodies to further define the role of specific CYP(s) (Rodrigues, 1999). Here,
enzyme kinetic parameters can also be obtained with pooled microsomal
preparations (multiple organ donors) or microsomes from individual
(genotyped) human liver preparations. Activity correlation studies are some-
times conducted to further elucidate the role of key CYP enzymes using a panel
of human liver microsomes from at least 10 different organ donors. In this
instance, metabolite formation is correlated with CYP form-specific activities
REACTION PHENOTYPING 127