Preliminary assessment of kinetics in human liver microsomes (Step 1)
revealed an S 50 of approximately 1 mM, which was the concentration used to
screen a panel of 12 commercially available recombinant UGTs (Step 2).
UGT1A3, UGT2B7, and UGT2B17 demonstrated potential to glucuronidate
gemcabene, and kinetics of glucuronidation was assessed using those individual
recombinant enzymes to assess similarities/differences in substrate affinity as a
potential differentiation factor (Step 3). No differentiation was achieved,
however, since the S 50 values were each approximately equivalent to human
liver microsomes 1 mM (Bauman et al., 2005).
Screening activities using candidate selective inhibitors of UGT1A3-,
UGT2B7-, or UGT2B17-catalyzed gemcabene glucuronidation revealed
5,6,7,3^0 ,4^0 ,5^0 -hexamethoxyflavone as an inhibitor of UGT1A3 and UGT2B17
(with four fold greater potency for recombinant UGT1A3) andS-flurbiprofen
as a selective UGT2B7 inhibitor (Step 4). Hexamethoxyflavone had no effect
on gemcabene glucuronidation in human liver microsomes, suggesting that
UGT1A3 and UGT2B17 do not significantly contribute to gemcabene
glucuronidation. Concordantly, the IC 50 forS-flurbiprofen inhibition of
gemcabene glucuronidation in human liver microsomes was similar to that
observed for recombinant UGT2B7, indicating that UGT2B7 was the major
contributor to gemcabene glucuronidation in human liver microsomes.
Further characterization of UGT2B7 as a major contributor to gemcabene
metabolism was achieved by using a phenotyped human bank of human liver
microsomes (Step 5): Rates of gemcabene glucuronidation correlated with
rates of glucuronidation of the UGT2B7 substrate zidovudine, but not with
rates of 3-glucuronidation of the UGT1A1 substrateb-estradiol (Bauman et
al., 2005).
The approaches described above (Bauman et al., 2005) present a resource-
intensive description of definitive UGT reaction phenotyping. Depending on
need, one or more of the described steps may be omitted. The area primed for
the greatest advance in the near future is the identification of selective
glucuronidation inhibitors. Note that competitive substrates for individual
enzymes are not necessarily selective inhibitors of those enzymes, since
compounds do not need to be substrates of a UGT enzyme to be an inhibitor of
that enzyme (Williams et al., 2002a). Recent advances have also been made in
in silico predictions (Sorich et al., 2002). This promising area should be
monitored for significant advances.
15.4.2 N-Acetylation Reaction Phenotyping
There are two human N-acetyltransferases, (NAT1 and NAT2). Substrates
of NAT1 include sulfamethoxazole (Nakamura et al., 1995), an ingredient of
the antibiotic combination BactrimTM, whereas isoniazid is a substrate of
NAT2 (Kinzig-Schippers et al., 2005). The polymorphic nature of theNAT1
gene is equivocal, whereas convincing evidence exists for functional
polymorphisms in theNAT2gene: genotype forNAT2explains 88% of the
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