for clearance through that pathway. In addition, attempts should be made to
identify the enzyme(s) responsible for formation of major circulating
metabolite(s) even though that pathway may be minor (<25%) in the overall
clearance of the compound. This is especially true for metabolites that may
have pharmacological or toxicological consequences.
There are numerous articles and reviews written on the conduct of the
reaction phenotyping studies using tools outlined above (Bjornsson et al., 2003;
Lu et al., 2003; Mei et al., 1999; Rodrigues, 1999; Williams et al., 2003; Zhang
et al., 2007). Chapter 15 provides additional information on the conduct of
the reaction phenotyping studies. In general, three methods for identifying the
individual CYP enzymes responsible for a metabolic pathway are typically used:
(1) incubation with individual human cDNA-expressed CYP enzymes, (2)
incubation with specific chemical inhibitors or monoclonal antibodies in pooled
human liver microsomes, and (3) incubation with a bank of human liver
microsomes that are prepared from individual donors and are well characterized
for CYP activities. At least two of the methods should be performed to identify
the specific enzyme(s) responsible for a metabolic pathway. If biotransformation
through a particular pathway is inhibited by a chemicals/monoclonal antibody
inhibitor and catalyzed by the expressed enzyme, then there is strong evidence
for the role of that enzyme in metabolism of the drug.
Human CYPs involved in metabolism can be easily characterized because
of the tools that are commercially available. However, similar tools are
not available for other drug-metabolizing enzymes like FMOs, UGTs, and
sulfotransferases. There are no specific inhibitors or monoclonal anitibodies
identified for any of the commercially available expressed UGTs or sulfo-
transferases. Due to the lack of necessary tools it is difficult to quantitatively
assess their contribution to the overall clearance of drugs that are metabolized
by these enzymes.
9.5.3 Evaluation of CYP Inhibition
A key set of studies that is conducted during discovery and development is to
understand the potential of a drug to inhibit different CYP enzymes. Potent
inhibition of CYPs may affect further development, specifically if they inhibit
key drug-metabolizing enzymes such as CYP1A2, CYP2A6, CYP3A4,
CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2D6. Evaluation of CYP
inhibition is described in detail in Chapters 5 and 16. A potential for CYP
inhibition can be evaluated either with human liver microsomes or with
expressed enzymes. A number of fluorescent substrates have been developed
for various CYPs (Cohen et al., 2003; Ghosal et al., 2003; Yamamoto et al.,
2002). These fluorescent substrates makes the assays amenable to high
throughput analysis. Such screens are usually used in the drug discovery
stage. Once a lead compound is identified for development then the inhibition
experiments are repeated in human liver microsomes to confirm the results
obtained from screening assays. FDA and EMEA usually consider inhibition
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