Hepatic cytosol is a useful source of sulfotransferases (Pacifici, 2004) and
aldehyde oxidase (Lake et al., 2002). Compared with cytochrome P450s, these
enzymes are rarely utilized in early drug metabolism screening and thus may be
limited to specific mechanistic studies or later stages of the drug development
process.
15.6.2.4 Whole-Cell Systems Hepatocytes offer the advantage over liver
microsomes of an integrated metabolic system, closer to thein vivosituation.
For prediction of metabolites formedin vivohepatocytes therefore offer a
useful system. For reaction phenotyping efforts the intact cell membrane
presents a barrier to entry into the cell for both the substrate and any selective
enzyme inhibitor that may be used. However, the sequential metabolism of
substrates that would be expected to occur in hepatocytes may not generate
sufficient clarity on which enzyme is the rate-limiting step determiningin vitro
clearance. Thus, subcellular fractions may be optimal systems for mechanistic
reaction phenotyping.
One significant advantage of hepatocytes over liver microsomes is that the
contribution of cytochrome P450 enzymes versus other cytosolic enzyme systems
can be assessed, using pan-cytochrome P450 inhibitors. Aminobenzotriazole is a
mechanism-based inhibitor of cytochrome P450 enzymes, and inhibits all of the
major cytochrome P450s in hepatocytes except CYP2C9 (using diclofenac as
substrate), without affecting glucuronidation or sulfation activities (Takahashi
and Bauman, Pfizer Global Research and Development, personal communica-
tion). Imidazole containing compounds such as sulconazole may be more
suitable compounds, since unlike aminobenzotriazole no preincubation is
required and CYP2C9 (using diclofenac as substrate) is also inhibited
(Takahashi and Bauman, Pfizer, personal communication).
15.6.3 Experimental Approach Considerations
This topic has been covered in some detail in a previous publication (Williams
et al., 2003), and the key points are summarized below.
15.6.3.1 Selection of Substrate and Enzyme Concentration Substrate
concentration is a key consideration for experimental design, since enzyme-
catalyzed metabolism is a saturable process, and the potential exists for differing
conclusions at different substrate concentrations when multiple enzymes
contribute toin vitroturnover. A good example is that of amitryptyline, where
in human liver microsomes CYP3A4 predominates at high concentrations,
whereas CYP2D6 predominates at low concentrations (Ghahramani et al.,
1997). In cases where the predicted therapeutic index of the compound is low, a
determination ofKmvalue (substrate concentration atVmax––half-maximal
reaction velocity) may be valuable in understanding the potential for nonlinear
pharmacokineticsin vivo. Compounds with very lowKm(e.g., below 1mM)
would have a higher chance of saturating the enzyme(s) responsible for clearance
NONRADIOLABELED REACTION PHENOTYPING 491