(e.g., NADPH). The concentration of solvents, particularly methanol and
DMSO, should be kept to a minimum (see Section 15.2). Reactions are
typically stopped by the addition of an ice-cold volume of ice-cold protein
precipitant (acetonitrile and trichloroacetic acid are common choices), and
spinning for precipitation will reduce the amount of unwanted protein injected
onto the HPLC column. Analysis of a compound is most easily achieved by
direct injection of an aliquot of the supernatant, rather than extraction, and the
former approach is most commonly used.
For substrate depletion approaches, the data can be plotted on a graph
(Fig. 15.4a) using simple visual inspection of the data. If only one recombinant
enzyme depletes parent drug, then it can be concluded that the enzyme in
question is the major contributor to metabolism in vitro(Figure 15.4a––
recombinant CYP1A2 is the only P450 contributing to parent depletion in this
example). If more than one enzyme appears to be depleting parent, then the
relative contributions of each can be estimated using a combination of the
slopes of (Fig 15.4b––both recombinant CYP1A2 and CYP2C9 are shown to
deplete parent drug over time), and the relative hepatic expression levels of the
metabolizing enzymes.
15.6.4.2 Chemical/Antibody Inhibition The selectivity of chemical inhibition
for cytochrome P450 inhibitors is often dependent on inhibitor concentration.
For example, ketoconazole is a potent and selective inhibitor of CYP3A
enzymes at 1mM concentration, whereas at higher concentrations it also
inhibits CYP2C8 and other enzymes. A list of selective chemical cytochrome
P450 inhibitors is shown in Table 15.1 (Tucker et al., 2001). Note that in
FIGURE 15.3 (Continued)NONRADIOLABELED REACTION PHENOTYPING 495