the mitochondial and microsomal subcellular fractions. Since the oxidative
enzymatic activities were highly correlated between the mitochondrial and
microsomal subcellular fractions in the Japanese livers, the microsomal
subcellular fractions were used for evaluation of the Caucasian livers. For
the chemical inhibition component of the study, selective inhibitors were added
5 min prior to addition of the substrate. Rizatriptan was primarily metabolized
by MAO-A and there were no apparent differences in MAO-A and MAO-B
activity between the Japanese and Caucasian livers.
15.3.3 Esterases
Historically, esterases have been classified according to either the compound
metabolized and/or the type of bond cleaved. However, this has changed as
technology has advanced regarding the molecular structure and function of
esterases, particularly for carboxylesterases (CES) and paraoxonases (PON)
(Li et al., 2005; Satoh et al., 2002). CES and cholinesterases both belong to a
protein super family designated by thea,b-hydrolase-fold family (Cygler et al.,
1993), while PON are unrelated to this protein super family (Draganov and
La Du, 2004; Satoh et al., 2002).
CES are the best-characterized esterases in regard to drug metabolism and
will be the focus of this section. Based on sequence homology, CES have been
classified into four families: CES1, CES2, CES3, and CES4 (Satoh and
Hosokawa, 1998). The two major human liver CES enzymes are hCE-1 and
hCE-2 that belong to the classes of CES1 and CES2, respectively (Satoh et al.,
2002). In mammals, CES is mainly localized in the endoplasmic reticulum of
many tissues (Satoh and Hosokawa, 1998; Zhu et al., 2000). Human liver S9 or
microsomes are usually used forin vitrostudies of CES, however, CES is also
located in cytosolic fractions (Tabata et al., 2004). In contrast to rat plasma,
human plasma contains no (or negligible) CES with esterase activity in human
plasma being due to cholinesterases, paroxonase, and/or serum albumin (Li
et al., 2005; McCracken et al., 1993). Phenotyping is limited for esterases due to
current limited availability of expressed esterases and commercially available
purified esterases. Reaction phenotyping for esterases is typically utilized in
mechanistic/descriptive studies for specific compounds/series rather than as a
broader screen in the drug discovery process like cytochrome P450 reaction
phenotyping.
Current examples of reaction phenotyping for CES include the studies
conducted describing the bioactivation of the anticancer agent irinotecan
(CPT-11, Camptosar)(Humerickhouse et al., 2000; Slatter et al., 1997) and the
metabolism of the prodrug capecitabine (Tabata et al., 2004). The bioactiva-
tion of irinotecan was evaluated by enzyme kinetic studies using human liver
microsomes and potent chemical inhibitors (carboxylesterase inhibitors:
physostigmine and bis-nitrophenyphosphate) (Slatter et al., 1997). The enzyme
kinetic data in human livers microsomes suggested that two enzymes catalyzed
the bioactivation of irinotecan (with low and high affinities). The metabolism
NONCYTOCHROME P450 REACTION PHENOTYPING 483