of irinotecan was further evaluated by enzyme kinetic studies using purified
hCE-1 and hCE-2 and bioactivation studies with purified hCE combined with
cytotoxicity studies (Humerickhouse et al., 2000). hCE-1 and hCE-2 were
identified as low and high affinity enzymes involved in the bioactivation of
irinotecan with theKmof hCE-2 nearly identical to theKmof the high affinity
enzyme observed previously in microsomes.
The enzyme responsible for the biotransformation of capecitabine to 5^0 -
deoxy-5-fluorocytidine (a precursor to 5-fluorouracil) was evaluated using
purified enzyme, cytosol, and microsomes. The purified CES cytosolic enzyme,
inhibited by the carboxylesterase inhibitors bis-nitrophenyphosphate and
diisopropylfluorophosphate, was identified as belonging to the subgroup
CES 1A1 based on the result of the N-terminal amino acid sequence.
15.4 Conjugation Phenotyping
15.4.1 UGT Reaction Phenotyping
The science behind conjugation reaction phenotyping has advanced more slowly
than cytochrome P450 phenotyping, due to the lower incidence and magnitude
of drug–drug interactions mediated by enzymes that catalyze conjugation
(Williams et al., 2004). Listed in decreasing order of occurrence for the top 200
drugs prescribed in the United States, glucuronidation, sulfation, and conjuga-
tion with amino acids or glutathione are possible pathways of metabolism of
drugs (Williams et al., 2004). Since amino acid conjugation is rarely a primary or
major pathway of metabolism for marketed drugs, reaction phenotyping of
glucuronidation catalyzed by UGTs, of sulfation catalyzed by sulfotransferases
(SULTs), and of N-acetylation catalyzed by N-acetyltransferases (NATs) will be
covered in this section. Targets for (soft) nucleophilic addition of sulfate or
glucuronide to drugs occur at similar sites, including hydroxy- and carboxylic
acid groups, and nucleophilic nitrogen and sulfur atoms.
Glucuronidation of parent drug is the most commonly listed conjugation
pathway of metabolism (Williams et al., 2004). It is important not to confuse
glucuronidation as (1) a primary clearance pathway of parent drug (sometimes
known as an aglycone), which is the topic of discussion here, with (2) a
secondary pathway of clearance of oxidative metabolites mostly catalyzed by
cytochrome P450 enzymes. Drugs that are cleared primarily by glucuronida-
tion include propofol, naloxone, zidovudine, and gemcabene (Bauman et al.,
2005). The UGT enzymes most commonly listed in catalyzing drug
glucuronidation are UGT1A1, UGT1A4, and UGT2B7 (Williams et al.,
2004).
As mentioned above, the primary contributors to pharmacokinetic variability
of metabolized drugs are clearance by polymorphic enzymes and drug–drug
interactions (Williams et al., 2003). However, the magnitude of drug–drug
interactions for glucuronidated drugs is typically low (Williams et al., 2004).
484 REACTION PHENOTYPING