expression levels of individual UGTs in human tissues and in individual
preparations of recombinant enzymes. To date, attempts to quantify protein
expression levels in human intestine and liver have not been successful.
Quantitative assessment of mRNA levels has been achieved, but the predictive
value of this information in anticipating protein levels is unproven. In addition
to knowledge of intestinal and hepatic expression levels, measures of relative
expression levels of UGTsin vitrowould context the quantitative contribution
of individual UGTs to the clearance of glucuronidated drugs. This is relatively
simple for preparations of recombinant P450 enzymes, in that nanomole
amounts of P450 per milligram protein can be measured by complexing the
P450 enzymes with carbon monoxide and reading absorbance at 450 nm. The
absence of validated methods for UGT enzyme quantitation, however, makes
measurements of nanomole enzyme per milligram protein for UGTs
challenging. The relative abundancein vitrotoin vivoextrapolation approach
(Proctor et al., 2004; Rodrigues, 1999) for predicting individual cytochrome
P450 enzyme contributions to clearance of P450 substrates could potentially be
applied to UGT substrates if the above two challenges of UGT protein
quantitation could be met.
A recent example of UGT reaction phenotyping is that for gemcabene, a
Pfizer compound indicated for treatment of dyslipidemia, and cleared primarily
via a single glucuronide in humans (Bauman et al., 2005). The strategy
employed for UGT reaction phenotyping compound is outlined in Fig. 15.2.
Although human liver microsomes were assayed in this example, the same
experimental approach could also be used for human intestinal microsomes. A
brief summary of the approach and key data follows.
FIGURE 15.2 Strategy for reaction phenotyping.486 REACTION PHENOTYPING