15.6.4.3 Correlation Analysis A human liver bank phenotyped for cyto-
chrome P450 activities (and potentially UGT activities) can provide further
confidence on the major pathways contributing toin vitroclearance. Although
more commonly associated with definitive reaction phenotyping where more
than 20 livers are used to assess rates of metabolism of the compound of
interest and compared to rates of metabolism, of the probe substrate (Hyland
et al., 2003), selection of a smaller number of livers (e.g., six, comprised of three
with high activity and three with low activity) in the earlier stages may also be
informative and could add further confidence. In the earlier stages of
development, correlation analysis is not commonly used. If resources are
constrained to a single approach, chemical inhibition is recommended.
15.6.4.4 Quantitative Reaction Phenotyping Using Metabolite Standard In
the presence of authentic standard of the major metabolite(s), it is first
necessary to develop an analytical assay using an appropriate detection method
(most commonly mass spectrometry). Secondly, it is important to define the
kinetics of metabolite formation using human liver microsomes. For reaction
phenotyping using authentic metabolite standard(s), an example of the utility
of recombinant enzymes and human liver microsomes with inhibitors is that of
sildenafil (Hyland et al., 2001). The major circulating metabolite for this
compound is formed via the N-demethylation of the piperazine ring.
Assessment of the kinetics of sildenafil N-demethylation in human liver
microsomes showed that at least two P450 enzymes were involved. At 250mM
sildenafil concentration N-demethylation was primarily mediated through a
low affinity highKm(81mM) enzyme, whereas at 2.5mM there was a greater
role for the high affinity, lowKm(6mM) enzyme (61%). Since ketoconazole
strongly inhibited metabolism at both substrate concentrations, it was
concluded that 75% or more of sildenafil N-demethylation activity is probably
attributable to CYP3A4 (Fig. 15.5a). Experiments with recombinant enzymes
indicated that CYP3A4 and to a lesser extent CYP2C9 contributed to sildenafil
N-demethylation (Fig. 15.5b). In addition, multivariate analysis of correlation
data across a human liver bank showed that the rate of sildenafil N-
demethylation strongly correlated with the rate of both CYP2C9 (phenytoin 4-
hydroxylation) and CYP3A4 (testosterone 6b-hydroxylation) mediated
metabolism.
15.6.5 Quantitative Reaction Phenotyping: Expressed or Purified Enzyme
Systems
Traditionally there are two approaches to quantitative reaction phenotyping
using either expressed or purified enzymes. One approach, the relative activity
factor (RAF) approach (Crespi and Miller, 1999; Stormer et al., 2000a;
Venkatakrishnan et al., 2001), is being increasingly used in estimating the
quantitative scaling of single enzyme data to multienzyme systems. The relative
activity factor is a methodology that normalizes the activity in expressed or
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