period, it will not be possible to define the relative contributions of each enzyme.
This presents a decision point on whether to advance the compound forward to
later stages in development with unknown enzymology, or to proceed with the
‘‘metabolite formation approach’’, which requires chromatography to separate
metabolites (Williams et al., 2005). In early stages, radiolabeled compounds and
authentic standards are not available, and thus peak responses should be
considered with care with regard to interpretation of relative amounts.
Quantitation of metabolites is challenging in the absence of radiolabel or
authentic standards. The most commonly used methods for metabolite
monitoring are detection by ultraviolet (UV) absorbance and by mass
spectrometry. When UV absorbance is being used for detection, the peak
response will be approximately proportional to the amount of metabolite if the
chromophore is not altered, such as hydroxylation of an aliphatic side group.
However, if the molecule is cleaved, altering conjugated systems, relative UV
peak responses may not accurately represent the relative amounts of parent
compound and each metabolite. Mass spectrometry is even less reliable than
UV absorbance in the absence of authentic standards, as ionization efficiency
for each detected molecule will differ. In practice, a combination of UV and
mass spectrometry is most appropriate. However, unlike the substrate
depletion approach, the possibility would still exist of ‘‘missing’’ metabolites
if the detection method does not pick up the metabolite of interest.
15.6.3.3 Reaction Velocity Linearity To achieve the most accurate assess-
ment of relative contributions of various enzymes to metabolism of a test
compound using the substrate depletion approach, the reaction must be
monitored where the rate of depletion of parent is proportional to its
concentration (a first order reaction). This justifies the selection of low
substrate concentrations (e.g., 1mM) in the incubation. This relationship
would appear as curved line using a linear scale (Fig. 15.3a) or a straight line
using a log-linear plot. For the metabolite formation approach, the generation
of metabolite would need to be linear with time and with protein, and would
need to be conducted under conditions where the concentration of parent
compound was not significantly (>15%) depleted. Figures 15.3b and c
respectively show examples of a compound where metabolite formation is
linear to 15 min incubation time (but not past that time period) and up to 1 mg/
mL protein (but not above that concentration). In general, reactions catalyzed
by cytochrome P450 enzymes do not maintain linearity past 45 min incubation
time. In contrast, reactions catalyzed by UGT enzymes can demonstrate
linearity of glucuronide production for up to 4 h.
15.6.4 Selection of Appropriate Experimental Designs
15.6.4.1 Expressed Enzymes In the early stages of drug development it is
appropriate to focus efforts on the five major cytochrome P450 enzymes
(CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5). For the purposes
NONRADIOLABELED REACTION PHENOTYPING 493